Antibody fc variants

ABSTRACT

The invention relates to engineered nolyneptides comprising Fc variants and their uses. More specifically, Fc variants are described exhibiting reduced effector function. These variantg cause a benefit for a patient suffering from a disease which could be treated with an antibody for which it is desirable to reduce the effector function elicited by antibodies.

This application is a divisional of U.S. patent application Ser. No.13/431,489 which claims the benefit of priority under 35 USC § 119 ofEuropean patent application number 11160251.2, filed on Mar. 29, 2011,each of which is hereby incorporated by reference in its entirety.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readablethrill (CRF) of the Sequence Listing (file name: 146392026510SEQLIST.txtdate recorded: May 6, 2015, size: 40 KB).

FIELD OF THE INVENTION

The present invention concerns polypeptides comprising variants of artFc region. More particularly, the present invention concerns Fcregion-containing polypeptides that have altered effector function as aconsequence of one or more amino acid substitutions in the Fc region ofthe polypeptide.

SUMMARY

The present invention relates to the field of antibody variants andprovides pdlypeptides comprising Fc variants with a decreased effectorfunction, like decreased ADCC andior C1q binding.

In particular the invention provides a polypeptide comprising an Fcvariant of a wild-type human IgG Fc region, said Fc variant comprisingan amino acid substitution at position Pro329 and at least one furtheramino acid substitution, wherein the residues are numbered according tothe EU index of Kabat, and wherein said polypeptide exhibits a reducedaffinity to the human FcγRIIIA and or FcγRIIA and/or FcγRI compared to apolypeptide comprising the wildtype IgG Fc region, and wherein the ADCCinduced by said polypeptide is reduced to at least 20% of the ADCCinduced by the polypeptide comprising a wild-type human IgG Fc region,

In a specific embodiment Pro 29 of a wild-type human Fc region in thepolypeptide described above is substituted with glycine arginine or anamino acid residue large enough to destroy the praline sandwich withinthe Fc/Fcγ receptor interface, that is formed between the proline 329 ofthe Fc and tryptopharte residues Trp 87 and Trp 110 of FcgRIII(Sondermann et at: Nature 406, 267-273 (20 Jul. 2000)). In a furtheraspect of the invention the at least one further amino acid substitutionin the Fc variant is S228F, E233P, L234A, L235A, L235E, N297A, N297D, orP331S and still in another embodiment said at least one further aminoacid substitution is L234A and L235A of the human IgG1 Fc region orS228P and L235E of the human Ig134 Fc region.

In another aspect of the invention the polypeptide provided exhibit; areduced affinity to at least one further receptor of the groupcomprising the human receptors FcγI, FcγIIA and C1q compared to thepolypeptide comprising a wild-type human IgG Fc region. In still anotheraspect of the invention the polypeptide CompriNes a human IgG1 or Ig64Fc region. In still another aspect of the invention the polypeptide isan antibody or an Fc fusion protein

In a further embodiment: the throinboeyte aggregation induced b thepolypeptide comprising the Fc variant is reduced compared to thethrombocyte aggregation induced by a polypeptide comprising a wild-typehuman IgG Fc region. In still a further embodiment, the pt peptideaccording to the invention exhibits a strongly reduced CDC compared tothe CDC induced by a polypeptide comprising a wild-type human IgG Fcregion.

In another embodiment of the invention polypeptides comprising an Fcvariant, as described above, are provided for use as a medicament, in aspecific embodiment the polypeptide is an anti-CD9 antibody, which ischaracterized in that the poll peptide comprising the wildtype Fc regioncomprises as heavy chain variable region SEQ ID NO:9 and as variablelight chain region SEQ. ID NO:8.

In another aspect of the invention the polypeptides as described aboveare provided for use in treating a disease wherein it is favorable thatan effector function of the polypeptide comprising the Fc variant isstrongly reduced compared to the effector function induced by apolypeptide comprising a wild-type human IgG Fc region,

In another embodiment the use of the polypeptides as described above isprovided for the manufacture of a medicament for the treatment: of adisease, wherein it is favorable that the effector function of thepolypeptide comprising an Fc variant of a wild-type human IgG Fc regionis strongly reduced compared to the effector function induced by apolypeptide comprising a wild-type human IgG Fc tegion.

In still another aspect of the invention a method of treating anindividual having a disease is provided, wherein it is favorable thatthe effector function of the polypeptide comprising an Fc variant of awild-type human IgG region is strongly reduced compared to the effectorfunction induced by a polypeptide comprising a wildtype human Fcpolypeptide, comprising administering to an individual an effectiveamount of the polypeptide described above.

A further aspect of the is is a use of a polypeptide comprising an Fcvariant of a wild-type human IgG Fc region, said polypeptide bayingPro329 of the human IgG Fc region substituted with glycine, wherein theresidues are numbered according to the EU index of Kabat, wherein saidpolypeptide exhibits a reduced affinity to the human Fc-fRITIA andFeyRTEA for down-modulation of ADCC to at least 20% of the ADCC inducedby the polypeptide comprising the wildlype human IgG Fc region, and/orfor down-modulation of ADCP.

Another aspect of the invention is use of a polypeptide cemprising an Fcvariant of a wild-type human IgG Fc rogion, said polypeptide havingPro329 of the human IgG Fc region substituted with gtyc nee and whereinthe Fc variant conifirises at least two further amino acid substitutionsat L234A and L235A of the human IgG1 Fc region or S228P and L235E of thehuman IgG4 Fc region, wherein the residues arc numbered according to theEU index of Kabat, wherein said pelypeptidc exhibits a reduced affinityto the human FcγRIIIA and FcγRIIA, for down-modulation of ADCC to atleast 20% of the ADCC induced by the polypeptide comprising the wildtypehuman IgG Fc region, and/or for down-modulation of ADCP.

Another aspect of the invention is use of thepolypeptide describedabove, wherein the thrombocyte aggregation in by the poiypeptidedescribed above is reduced compared to the thrombocyte aggregationinduced by a polypeptide comprising a wildtype human Fc region, whereinthe polypeptide is a platelet activating antibody.

In another aspect of the invention a method of treating an individualhaving a disease is provided, wherein said individual is treated with apolyneritide, said polypeptide having Pro329 of the human IgG Fc regionsubstituted with glycine, wherein the residues are numbered according tothe EU index of Kabat, wherein said polypeptide characterized by astrongly reduced binding FcγRIIIA and/or FcγRIIA compared to apolypeptide comprising a wildtype human IgG Fc region, comprisingadministering to the individual an effective amount of said polypeptide.

In still another aspect of the invention the polypeptide used in saidmethod nomprises at least two further amino acid substitutions at L234Aand L235A of the human IgG1 Fc region or S228P and L235E of the humanIgG4 Fc region.

BACKGROUND

Monoolonal antibodies have great therapeutic potential and play animportant role in today's medical portfolio. During the last decade, asignificant trend in the pharmaceutical industry has been thedevelopment of monoclonal antibodies. (mAbs) as therapeutic agents forthe treatment of a number of diseases, such as cancers, asthma,arthritis, multiple sclerosis etc. Monoclonal antibodies arepredominantly manufactured as recombinant proteins in geneticallyengineered mammalian cell culture.

The Fc region of an antibody, i.e., the terminal ends of the heavychains of antibody spanning domains CH2, CH3 and a portion of the hingeregion, is limited in variability and is involved in effecting thephysiological roles played by the antibody. The effector functionsattributable to the Fc region of an antibody vary with the class andsubclass of antibody and include binding of the antibody via the Fcregion to a specific Fc receptor (“FcR”) on a cell which triggersvarious biological responses,

These receptors typically have an extracellular domain that mediatesbinding to Fc, a membrane spanning region, and an intracellular domainthat may mediate some signaling event within the cell. These receptorsare expressed in a variety of immune cella including monocytes,macrophages, neutrophils, dendritic cells, cosinophils, mast cells,platelets, B cells, large granular lymphocytes, Langerhans' cells,natural killer (NK) cells, and T cells. Formation of the Fc/FcγR complexrecruits these effector cells to sites of bound antigen, typicallyresulting in signaling events within the cells and important subsequentimmune responses such as release of inflammation mediators, B cellactivation, endocytosis, phagocytosis, and cytotoxic attack. The abilityto mediate cytotoxic and phagocytic effector functions is a potentialmechanism by which antibodies destroy targeted cells, The cell-mediatedreaction wherein nonspecific cytotoxic cells that express FcγRsrecognize bound antibody on a target cell and subsequently cause lysisof the target cell is referred to as antibody dependent cell-mediatedcytotoxicity (ADCC) (Ravetch, et al., Annu Rev Immunol 19 (2001)275-290), The cell-mediated reaction wherein nonspecific cytotoxic cellsthat express FcγRs recognize bound antibody on a target cell andsubsequently cause phagocytosis of the target cell is referred to asantibody dependent cell-mediated phagocytosis (ADCP). In addition, anoverlapping site on the Fc region of the molecule also controls theactivation of a cell independent cytotoxic function mediated bycomplement, otherwise known as complement dependent cytotoxicity (CDC).

For the IgG class of Abs, ADCC and ADCP are governed by engagement ofthe Fc region with a family of receptors referred to as Fcγ receptors(FcγRs). In humans, this protein family comprises FcγRI (CD64); FcγRII(CD32), including isoforms FcγRIIA, FcγRIIB, and FcγRIIC; and FcγRIII(CD16), including isoforms FcγRIIIA and FcγRIIIB (Raghavan, andBjorkman, Annu. Rev. Cell. Dev. Biol. 12 (1996) 181-220; Abes, et al.,Expert Reviews VOL 5(6), (2009) 735-747). FcγRs are expressed on avariety of inmune cells, and formation of the Fc/FcγR complex recruitsthese cells to sites of bound antigen, typically resulting in signalingand subsequent immune responses such as release of inflammationmediators, B cell activation, endocytosis, phagocytosis, and cytotoxicattack. Furthermore, whereas FcγRI, FcγRIIA/c, and FcγRIIIA areactivating receptors characterized by an intracellular immunoreceptortyrosine-based activation motif (ITAM), FcγRIIB has an inhibition motif(ITIM) and is therefore inhibitory. Moreover, de Reys, et al., Blood,81, (1993) 1792-1800 concluded that platelet activation and aggregationinduced by monockemal antibodies, like for example CD9, is initiated byantigen recognition followed by an Fc domain dependent step, whichinvolves the FcγRII-receptor (see also: Taylor, et al., Blood 96 (2000)4254-4260). While FcγRI binds monomeric IgG with high affinity, FcγRIIIand FcγRII are low-affinity receptors. interacting with complexed oraggregated IgG.

The complement inflammatory cascade is a part of the innate immuneresponse and is crucial to the ability for an individual to ward offinfection. Another important Fe ligand is the complement protein C1q Fcbinding to C1q mediates a process called complement dependentcytotoxicity (CDC). C1q is capable of binding six antibodies, althoughbinding to two IgGs is sufficient to activate the complement cascade,C1q forms a complex with the C1r and C1s serine proteases to form the C1complex of the complement pathway.

In many circumstances, the binding and stimulation of effector functionsmediated by the Fc region of immunoglobulins is highly beneficial. e.g.for a CD20 antibody. however, in certain instances it may be moreadvantageous to decrease or even to eliminate the effector fonction.This is particularly true for those antibodies designed to deliver adrug (e.g., toxins and isotopes) to the target cell where the Fc/FcγRmediated effector functions bring healthy immune cells into theproximity of the deadly payload, resulting in depletion of normallymphoid tissue along with the target cells (Hutchins, et al., PNAS USA92 (1995) 11980-11984; White, et al., Annu Rev Med 52 (2001) 125-145),In these cases the use of antibodies that poorly recruit complement oreffector cells would be of a tremendous benefit (see also, Wa, et al.,Cell Immunol 200 (2000) 16-26; Shields, et al., J. Biol Chem 276(9)(2001) 6591-6604: U.S. Pat. Nos. 6,194,551; 5,885,573 and PCTpublication WO 04/029207).

In other instances, for example, where blocking the interaction of awidely expressed receptor with its cognate ligand is the objective, itwould be advantageous to decrease or eliminate all antibody effectorfunction to reduce unwanted toxicity, Also, in the instance where atherapeutic antibody exhibited promiscuous binding across a number ofhuman tissues it would be prudent to limit the targeting of effectorfunction to a diverse set of tissues to limit toxicity. Last but notleast, reduced affinity of antibodies to the FcγRII receptor inparticular would be advantageous for antibodies inducing plateletactivation and aggregation via FcγRII receptor binding, which would be aserious side-effect of such antibodies.

Although there are certain subclasses of human immunoglobulins that lackspecific effector functions, there are no known naturally occurringimmunoglobulins that lack all effector functions. An alternate approachwould be to engineer or mutate the critical residues in the Fc regionthat are responsible for effector function. For examples see PCTpublications WO 2009/100309 (Medinimune), WO 2006/076594 (Xencor), WO1999/58572 (Univ. Cambridge), US 2006/0134709 (Macrogenics), WO2006/047350 (Xencor), WO 2006/053301 (Xencor), U.S. Pat. Nos. 6,737,056(Genentech), 5,624,821 (Scotgen Pharmaceuticals), and US 2010/0166740(Roche).

The binding of IgG to activating and inhibitory Fcγreceptors or thefirst component of complement (C1q) depends on residues located in thehinge region and the CH2 domain. Two regions of the CH2 domain arecritical for FcγRs and complement C1q binding, and have uniquesequences. Substitution of human IgG1 and IgG2 residues at positions233-236 and IgG4 residues at positions 327, 330 and 331 greatly reducedADCC and CDC (Armour, et al., Eur. J. Immunol. 29(8) (1999) 2613-2624;Shields, et al., J. Biol. Chem. 276(9) (2001) 6591-6604), Idusogie, etal., J. Immunol 166 (2000) 2571-2575) mapped the C1q binding site forrituxan and showed that Pro329Ala reduced the ability of Rituximab tobind C1q and activate complement. Substitution of Pro329 with Ala hasbeen reported to lead to a reduced binding to the FcγRI FcγRII andFcγRIIIA receptors (Shields, et al., J. Biol. Chem. 276(9) (2001)6591-6604) but this mutation has also been described as exhibiting awildtype-like binding to the FcγRI and FcγRII and only a very smalldecrease in binding to the FcγRIHIA receptor (Table 1 and Table 2 in EP1 068 241, Genentech).

Oganesyan, et al., Acta Cristallographica D64 (2008) 700-704 introducedthe triple mutation L234F/L23SE/P331S into the lower hinge and C2Hdomain and showed a decrease in binding activity to human IgG1 moleculesto human C1q receptor, FcγRI, FcγRII and FcγRIIIA.

Still, there is an unmet need for antibodies with a strongly decreasedADCC and/or ADCP and/or CDC. Therefore, the aim of the current inventionwas to identify such antibodies. Surprisingly, it has been found thatmutating the proline residue at Pro329 to glycine resulted in anunexpected strong inhibition of the FcγRIIIA and FcγRIIA receptor and ina strong inhibition of ADCC and CDC. Moreover, the combined mutation ofPro329 and for example L234A and L235A (LALA) lead to an unexpectedstrong inhibition of C1q, FcγRI, FcγRII and FcγRIIIA. Thus, a glycineresidue appears to be unexpectedly superior over other amino acidsubstitutions, like alanine, for example, at position 329 in destroyingthe proline sandwich in the Fc/Fcγ receptor interlace.

DESCRIPTION OF THE FIGURES

FIGS. 1 a-1 e

Binding affinities of different FcγRs towards immunoglobulins weremeasured by Surface Plasmon Resonance (SPR) using a Biacore T100instrument (GE Healthcare) at 25° C.

-   -   FIG. 1 a ) FcγRI binding affinity was tested for GA101 (GA)        antibody variants (IgG1-P329G, IgG4-SPLE and IgG1-LALA mutation)        and for P-selectin (PS) antibody variants (IgG1-P329G, IgG1-LALA        and IgG4-SPLE) as well as for the wildtype antibodies.    -   FIG. 1 b ) FcγRI binding affinity was tested for CD9 antibody        variants (IgG1-wildtype, IgG1-P329G, IgG1-LALA, IgG4-SPLE,        IgG1-P329G/LALA, IgG4-SPLE/P329G) as well as for the wildtype        antibodies.    -   FIG. 1 c ) FcγRIIA(R131) binding affinity was tested for CD9        antibody variants (IgG1-wildtype, IgG1-P329G, IgG1-LALA,        IgG4-SPLE, IgG1-P329G/LALA, IgG4-SPLE/P329G) as well as for the        wildtype antibodies. A normalized response is shown as a        function of the concentration of the receptor.    -   FIG. 1 d ) FcγRJIB binding affinity was tested for CD9 (named        here:“TA”) antibody variants (IgG1-wildtype, IgG4-SPLE/P329G,        IgG1-LALA, IgG1-LALA/P329G) and P-selectin (pSel) antibody        variants (IgG4-wildtype, IgG4-SPLE) as well as for the wildtype        antibodies.    -   FIG. 1 e ) FcγRIIIA-V158 binding affinity was tested for CD9        antibody variants (IgG1-wildtype, IgG4-SPLE, IgG1-LALA,        IgG4-SPLE/P329G, IgG1-P329G, IgG1-LALA/P329G) as well as for the        wildtype antibodies. a normalized response is shown as a        function of the concentration of the receptor.

FIG. 2

C1q binding was tested for P-selectin (PS) antibody variants (IgG1wildtype, P329G, IgG4-SPLE) and CD20 (GA) antibody variants(IgG1-wildtype, P329G and IgG4-SPLE).

FIGS. 3 a and 3 b

Potency to recruit immune-effector cells depends on type of Fc variant.Fc variants were coated on an ELISA plate and human NK92 effector cellstransfected with human FcγRIIIA were added. Induction of cytolyticactivity of activated NK cells was measured using an esterase assay.

-   -   FIG. 3 a ) CD20 (GA101) antibody variants (wildtype, LALA,        P329G, P329G /LALA) were analyzed. FIG. 3 b ) CD20 (GA101)        antibody variants (P329R or P329G mutations introduced) were        analyzed. . All variants were produced in the glycoengineered        version in order to have a stronger signal for any effector cell        recruitment function.

FIGS. 4 a and 4 b

Potency to recruit immune-effector cells depends on type of Fc variant,as measured by classical ADCC assay. Human NK92 cell-line transfectedwith human PγcRIIIA was used as effector and CD20 positive Raji cellswere used as target cells. Different glycengineered CD20 antibody (GA101G(2) and non-glycoengineered CD20 antibody (GA101) variants (P329G,P329A or LALA mutations introduced) were tested.

-   -   FIG. 4 a ) non-glycoengineered CD20 antibody : P329G, LALA and        P329G/LALA mutations, respectively, have been introduced into        the antibody, respectively.    -   FIG. 4 b ) glycoengineered CD20 antibody: P329G, P329A and LALA        mutations, respectively, have been introduced into the antibody        , respectively.

FIGS. 5 a and 5b

Complement dependent cytotoxicity (CDC) assay. The different Fc variantsof a non-glycoengineered and glycoengineered CD20 (GA101) antibody wereanalyzed for their efficacy to mediate CDC on SUDH-L4 target cells.

-   -   FIG. 5 a ) non-glycoengineered CD20: P329G, LALA and P329G/LALA        mutations, respectively, have been introduced into the antibody,        respectively.    -   FIG. 5 b ) glycoengineered CD20: P329G, P329A and LALA        mutations, respectively, have been introduced into the antibody,        respectively.

FIGS. 6 a and 6 b

-   -   FIG. 6 a ) Carbohydrate profile of Fc-associated glycans of        human IgG1 variants. The percentage of galactosylation on        Fc-associated oligosacchrides of hIgG1 containing the LALA,        P329G, P329A or P329G/LALA mutations only differs minimally from        that of wild type antibody.    -   FIG. 6 b ) Relative galactosylation: Four different IgGs with        introduced IgG1 P329G/LALA mutations. Four different V-domains        were compared for their amount of galactosylation when expressed        in Hek293 EBNA cells.

FIGS. 7 a and 7 b

Antibody-induced platelet aggregation in whole blood assay. Murine IgG1induced platelet aggregation as determined for two donors differing intheir response in dependence of the antibody concentration.

FIG. 7 a ) Donor A, FIG. 7 b ) Donor B.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

In the present specification and claims, the numbering of the residuesin an immunoglobulin heavy chain is that of the EU index as in Kabat, etal., Sequences of Proteins of Immunological Interest, Sth Ed. PublicHealth Service, National Institutes of Health, Bethesda, Md. (1991),expressly incorporated herein by reference. The “EU index as in Kabat”refers to the residue numbering of the human IgG1 EU antibody.

“Affinity” refers to the strength of the sum total of concovalentinteractions between a single binding site of a molecule (c.g., anantihody) and its binding partner (e.g., an antigen or an Fc receptor).Unless indicated otherwise, as used herein, “binding affinity” refers tointrinsic binding affinity which reflects a 1:1 interaction betweenmembers of a binding pair (e.g., antibody/Fc receptor or antibody andantigen). The affinity of a molecule X for its partner Y can generallybe represented by the dissociation constant (Kd). Affinity can hemeasured by common methods known in the art, including those describedherein. Specific illustrative and exemplary embodiments for measuringbinding affinity are described in the following.

An “affinity matured” antibody refers to an antibody with one or morealterations in one or more hypervariable regions (HVRs), compared to aparent antibody which does not possess such alterations, suchalterations resulting in an improvement in the affinity of the antibodyfor antigen.

An “amino acid modification” refers to a change in the amino acidsequence of a predetermined amino acid sequence. Exemplary modificationsinclude an amino acid substitution, insertion and/or deletion. Thepreferred amino acid modification herein is a substitution. An “aminoacid modification at” a specified position, e.g. of the Fc region,refers to the substitution or deletion of the specified residuo, or theinsertion of at least one amino acid residue adjacent the specifiedresidue. By insertion “adjacent” a specified residue is meant insertionwithin one to two residues thereof. The insertion may be N-terminal orC-terminal to the specified residue.

An “amino acid substitution” refers to the replacement of at least oneexisting amino acid residue in a predetermined amino acid sequence withanother different “replacement” amino acid residue. The replacementresidue or residues may be “naturally occurring amino acid residues”(i.e. encoded by the genetic code) and selected from the groupconsisting of: alanine (Ala): arginine (Arg); asparagine (Asn); asparticacid (Asp); cysteine (Cys); glutamine (Gln); glutamic acid (Glo):glycine (Gly); histidine (His); isoleucine (Ile): leucine (Leu); lysine(Lys); methionine (Met); phenylalanine (Phe); proline (Pro); serine(Ser); threonine (The); tryptophan (Trp); tyrosine (Tyr); and valine(Val), Preferably, the replacement residue is not cysteine. Substitutionwith one or more non-naturally occurring amino acid residues is alsoencompassed by the definition of an amino acid substitution herein. A“non-naturally occurring amino acid residue” refers to a residue, otherthan those naturally occurring amino acid residues listed above, whichis able to covalently bind adjacent amino acid residues(s) in apolypeptide chain. Examples of non-naturally occurring amino acidresidues include norleucine, ornithine, norvaline, homoscrine and otheramino acid residue analogues such as those described in Ellman, et al.,Meth. Enzym. 202 (1991) 301-336. To generate such non-naturallyoccurring amino acid residues, the procedures of Noren, et al., Science244 (1989) 182 and Ellman, et al., supra, can be used. Briefly, theseprocedures involve chemically activating a suppressor IRNA with anon-naturally occurring amino acid residue followed by in vitrotranscription and translation of the RNA.

An “amino acid insertion” refers to the incorporation of at least oneamino acid into a predetermined amino acid sequence. While the insertionwill usually consist of the insertion of one or two amino acid residues,the present application contemplates larger “peptide insertions”, e.g.insertion of about three to about five or even up to about ten aminoacid residues. The inserted residue(s) may be naturally occurring ornon-naturally occurring as disclosed above.

An “amino acid deletion” refers to the removal of at least one aminoacid residue from a predetermined amino acid sequence.

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, moltispecific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as they exhibitthe desired antigen-binding activity.

The term “antibody variant” as used herein refers to a variant of awildtype antibody, characterized in that an alteration in the amino acidsequence relative to the wildtype antibody occurs in the antibodyvariant, e.g. introduced by mutations a specific amino acid residues inthe wildtype antibody.

The term “antibody effector function(s),” or “effector function” as usedherein refers to a function contributed by an Fc effector domain(s) ofan IgG (e.g., the Fc region of an immunoglobulin). Such function can beeffected by, for example, binding of an Fc effector domain(s) to an Fcreceptor on an immune cell with phagocytic or lytic activity or hybinding of an Fc effector domain(s) to components of the complementsystem. Typical effector functions are ADCC, ADCP and CDC.

An “antibody fragment” refers to a molecule other than an intactantibody that comprises a portion of an intact antibody that binds theantigen to which the intact antibody binds. Examples of antibodyfragments include but are not limited to Fv, Fab, Fab′, Fab′-SH,F(ab′)₂; diabodies; linear antibodies; single-chain antibody molecules(e.g. scFv); and multispecific antibodies formed from antibodyfragments.

An “antibody that binds to the same epitope” as a reference antibodyrefers to an antibody that blocks binding of the reference antibody toits antigen in a competition assay by 50% or more, and conversely, thereference antibody blocks binding of the antibody to its antigen in acompetition assay by 50% or more. An exemplary competition assay isprovided herein.

“Antibody-dependent cell-mediated cytotoxicity” and “ADCC” refer to acell-mediated reaction in which nonspecific cytotoxic cells that expressFcRs (e.g. Natural Killer (NK) cells, neutrophils, and macrophages)recognize bound antibody on a target cell and subsequently cause lysisof the target cell. The primary cells for mediating ADCC, NK cells,express FcγRIII only, whereas monocytes express FcγRI, FcγRII andFcγRIII. FcR expression on hematopoietic cells is summarized in Table 3on page 464 of Ravetch, and Kinet, Annu. Rev. Immunol 9 (1991) 457-492.

The term “Antibody-dependent cellular phagocytosis” and “ADCP” refer toa process by which antibody-coated cells are internalized, either inwhole or in part, by phagocytic immune cells (e.g., macrophages,neutrophils and dendritic cells) that bind to an immunoglobulin Fcregion.

The term “binding domain” refers to the region of a polypeptide thatbinds to another molecule. In the case of an FcR, the binding domain cancomprise a portion of a polypeptide chain thereof (e.g. the α chainthereof) which is responsible for binding an Fc region. One usefulbinding domain is the extracellular domain of an FcR α chain.

The term “binding” to an Fc receptor used herein means the binding ofthe antibody to a Fc receptor in a BIAcore(R) assay for example(Pharmacia Biosensor AB, Uppsala, Sweden).

In the BIAcore(R) assay the Fc receptor is bound to a surface andbinding of the variant, e.g. the antibody variant to which mutationshave been introduced, is measured by Surface Plasmon Resonance (SPR).The affinity of the binding is defined by the terms ka (rate constantfor the association of the antibody from the antibody/Fc receptorcomplex), kd (dissociation constant), and KD (kd/ka). Alternatively, thebinding signal of a SPR sensogram can be compared directly to theresponse signal of a reference, with respect to the resonance signalheight and the dissociation behaviors.

“C1q” is a polypeptide that includes a binding site for the Fc region ofan immunoglobulin. C1q together with two serine proteases, C1r and C1s,forms the complex C1, the first component of the complement dependentcytotoxicity (CDC) pathway. Human C1q can be purchased commerciallyfrom, e.g. Quidel, San Diego, Calif.

The “CH2 domain” of a human IgG Fc region (also referred to as “Cy2”domain) usually extends from about amino acid 231 to about amino acid340, The CH2 domain is unique in that it is not closely paired withanother domain. Rather, two N-linked branched carbohydrate chains areinterposed between the two CH2 domains of an intact native IgG molecule.It has been speculated that the carbohydrate may provide a substitutefor the domain-domain pairing and help stabilize the CH2 domain (Burton,Molec. Immunol. 22 (1985) 161-206).

The “CH3 domain” comprises the stretch of residues C-terminal to a CH2domain in an Fc region (i.c. from about amino acid residue 341 to aboutamino acid residue 447 of an IgG).

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include but are not limitedto, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. Moreparticular examples of such cancers include squamous cell cancer,small-cell lung cancer, non-small cell lung cancer, adenocarcinoma ofthe lung, squamous carcinoma of the lang, cancer of the peritoneum,hepatocellular cancer, gastrointestinal cancer, pancreatic cancer,glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladdercancer, hepatoma, breast cancer, colon cancer, colorectal cancer,endometrial or uterine carcinoma, salivary gland carcinoma, kidneycancer. liver cancer, prostate cancer, vulval cancer, thyroid cancer,hepatic carcinoma and various types of head and neck cancer.

As used herein, the expressions “cell,” “cell line,” and “cell culture”are used interchangeably and all such designations include progeny.Thus, the words “transformants” and “transformed cells” include theprimary subject cell and cultures derived there from without regard forthe number of transfers. It is also understood that all progeny may notbe precisely identical in DNA content, due to deliberate or inadvertentmutations. Mutant progeny that have the same function or biologicalactivity as screened for in the originally transformed cell areincluded. Where distinct designations are intended, it will be clearfrom the context.

The term “chimeric” antibody refers to an antibody in which a portion ofthe heavy and/or light chain is derived from a particular source orspecies, while the remainder of the heavy and/or light chain is derivedfrom a different source of species.

The “class” of an antibody refers to the type of constant domain orconstant region possessed by its heavy chain. There are five majorclasses of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of thesemay be further divided into subclasses (isotypes), e.g., IgG₁, IgG₂,IgG₃, IgG₄, IgA₁, and IgA₂. The heavy chain constant domains thatcorrespond to the different classes of immunoglobulins are called α, β,ϵ, γ and μ, respectively.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents a cellular function and/or causes cell death ordestruction. Cytotoxic agents include, but are not limited to,radioactive isotopes (e.g., At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³,Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu); chemotherapeuticagents or drugs (e.g., methotrexate, adriamicin, vinca alkaloids(vincristine, vinblastine, ctoposide), doxorubicin, melphalan, mitomycinC, chlorambucil, daunorubicin or other intercalating agents); growthinhibitory agents; enzymes and fragments thereof such as nucleolyticenzymes; antibiotics; toxins such as small molecule toxins orenzymatically active toxins of bacterial, fungal, plant or animalorigin, including fragments and/or variants thereof; and the variousantitumor or anticancer agents disclosed below.

The term “complement-dependent cytotoxicity” or CDC refers to amechanism for inducing cell death in which an Fc effector domain(s) of atarget-bound antibody activates a series of enzymatic reactionsculminating in the formation of holes in the target cell membrane.Typically, antigen-antibody complexes such as those on antibody-coatedtarget cells bind and activate complement component C1q which in turnactivates the complement cascade leading to target cell death.Activation of complement may also result in deposition of complementcomponents on the target cell surface that facilitate ADCC by bindingcomplement receptors (e.g., CR3) on leukocytes.

A “disorder” is any condition that would benefit from treatment with apolypeptide. like antibodies comprising an Fc variant. This includeschronic and acute disorders or diseases including those pathologicalconditions which predispose the mammal to the disorder in question. Inone embodiment, the disorder is cancer.

“Effector functions” refer to those biological activities attributableto the Fc region of an antibody, which vary with the antibody isotype.Examples of antibody effector functions include: C1q binding andcomplement dependent cytotoxicity (CDC): Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis(ADCP); down regulation of cell surface receptors (e.g. B cellreceptor); and B cell activation.

A “reduced effector function” as used herein refers to a reduction of aspecific effector function, like for example ADCC or CDC, in comparisonto a control (for example a polypeptide with a wildtype Fc region), byat least 20% and a “strongly reduced effector fonction” as used hereinrefers to a reduction of a specific effector function, like for exampleADCC or CDC, in comparison to a control, by at least 50%.

An “effective amount” of an agent, e.g., a pharmaceutical formulation,refers to an amount effective, at dosages and for periods of timenecessary, to achieve the desired therapeutic or prophylactic result.

The term “Fc region” herein is used to define a C-terminal region of animmunoglobulin heavy chain that contains at least a portion of theconstant region. The term inchides native sequence Fc regions andvariant Fc regions. In one embodiment, a human IgG heavy chain Fc regionextends from Cys226, or from Pro230, to the carboxyl-terminos of theheavy chain. However, the C-terminal lysine (Lys447) of the Fc regionmay or may not be present. Unless otherwise specified herein, numberingof amino acid residues in the Fc region or constant region is accordingto the EU numbering system, also called the EU index, as described inKabat, et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, MD(1991).

A “variant Fc region” comprises an amino acid sequence which differsfrom that of a “native” or “wildtype” sequence Fc region by virtue of atleast one “amino acid modification” as herein defined. Preferably, thevariant Fc region has at least one amino acid substitution compared to anative sequence Fc region or to the Fc region of a parent polypeptide,e.g. from about one to about ten amino acid substitutions, andpreferably from about one to about five amino acid substitutions in anative sequence Fc region or in the Fc region of the parent polypeptide.The variant Fc region herein will preferably possess at least about 80%homology with a native sequence Fc region and/or with an Fc region of aparent polypeptide, and most preferably at least about 90% homologytherewith, more preferably at least about 95% homology therewith.

The term “Fc-variant” as used herein refers to a polypeptide comprisinga modification in an Fc domain. The Fc variants of the present inventionare defined according to the amino acid modifications that compose them.Thus, for example. P329G is an Fc variant with the substitution ofproline with glycine at position 329 relative to the parent Fcpolypeptide, wherein the numbering is according to the EU index. Theidentity of the wildtype amino acid may be unspecified, in which casethe aforementioned variant is referred to as P329G. For all positionsdiscussed in the present invention, numbering is according to the EUindex. The EU index of EU index as in Kabat or EU numbering schemerefers to the numbering of the EU antibody (Edelman, et al., Proc NatlAcad Sci USA 63 (1969) 78-85, hereby entirely incorporated byreference.) The modification can be an addition, deletion, orsubstitution. Substitutions can include naturally occurring amino acidsand non-naturally occurring amino acids. Variants may comprisenon-natural amino acids. Examples include U.S. Pat. No. 6,586,207; WO98/48032; WO 03/073238; US 2004/0214988 A1; WO 05/35727 A2; WO 05/74524A2; Chin, J. W., et al., Journal of the American Chemical Society 124(2002) 9026-9027; Chin, J. W., and Schultz, P. G., ChemBioChem 11 (2002)1135-1137; Chin, J. W., et al., PICAS United States of America 99 (2002)11020-11024; and, Wang, L., and Schultz, P. G., Chem. (2002) 1-10, allentirely incorporated by reference.

The term “Fc region-containing polypeptide” refers to a polypeptide,such as an antibody or immunoadhesin (see definitions below), whichcomprises an Fc region.

The terms “Fc receptor” or “FcR” are used to describe a receptor thatbinds to the Fc region of an antibody. The preferred FcR is a nativesequence human FcR. Moreover, a preferred FcR is one which binds an IgGantibody (a gamma receptor) and includes receptors of the FcγRI, FcγRII,and FcγRIII subclasses, including allelic variants and alternativelyspliced forms of these receptors. FcγRII receptors include FcγRIIA (an“activating receptor”) and FcγRIIB (an “inhibiting receptor”), whichhave similar amino acid sequences that differ primarily in thecytoplasmic domains thereof, Activating receptor FcγRIIA contains animmunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmicdomain. Inhibiting receptor FcγRIIB contains an immunoreceptortyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (seereview in Daeron, M., Annu. Rev. Immunol. 15 (1997) 203-234). FcRs arereviewed in Ravetch, and Kinet, Annu. Rev. Immunol 9 (1991) 457-492;Capel, et al., Immunomethods 4 (1994) 25-34; and de Haas, et al., J.Lab, Clin. Med. 126 (1995) 330-41. Other FcRs, including those to beidentified in the future, are encompassed by the term “FcR” herein. Theterm also includes the neonatal receptor, FcRn, which is responsible forthe transfer of maternal IgGs to the fetus (Guyer, et al., J. Immunol.117 (1976) 587 and Kim, et al., J. Immunol. 24 (1994) 249).

By “IgG Fc ligand” as used herein is meant a molecule, preferably apolypeptide, from any organism that binds to the Fc region of an IgGantibody to form an Fc/Fc ligand complex. Fc ligands include but are notlimited to FcγRs, FcγRs, FcγRs, FcRn, C1q, C3, mannan binding lectin,mannose receptor, staphylococcal protein A. streptococcal protein G, andviral FcγR, Fc ligands also include Fc receptor homologs (FcRH), whichare a family of Fc receptors that are homologous to the FcγRs (Davis, etal., Immunological Reviews 190 (2002) 123-136, entirely incorporated byreference). Fc ligands may include undiscovered molecules that bind Fc.Particular IgG Fc ligands are FcRn and Fc gamma receptors. By “Fcligand” as used herein is meant a molecule, preferably a polypeptide,from any organism that binds to the Fc region of an antibody to form anFc/Fc ligand complex.

By “Fc gamma receptor”, “FcγR” or “FogammaR” as used herein is meant anymember of the family of proteins that bind the IgG antibody Fc regionand is encoded by an PcyR gene. In humans this family includes but isnot limited to FcγRI (CD64), including isoforms FcγRIA, FcγRIB, andFcγRIC; FcγRII (CD32), including isoforms FcγRILA (including allotypesH131 and R131), FcγRIB (including FcγRIIB-1 and FcγRHIB-2), and FcγRIle;and FcγRIII (CD 16), including isoforms FcγRIHIA (including allotypesV158 and F158) and FcγRIIIb (including allotypes FoyRIIB-NA1 andFcγRIIB-NA2) (Jefferis, et al., Immunol Lett 82 (2002) 57-65, entirelyincorporated by reference), as well as any undiscovered human FcγRs orFcγR isoforms or allotypes. An FcγR may be from any organism, includingbut not limited to homans, mice, rats, rabbits, and monkeys. Mouse FcγRsincinde but are not limited to FcγRI (CD64), FcγRII (CD32), FcγRIII(CD16), and FcγRIII-2 (CD16-2), as well as any undiscovered mouse FcγRsor FcγR isoforms or allotypes.

By “FcRn” or “neonatal Fc Receptor” as used herein is meant a proteinthat binds the IgG antibody Fc region and is encoded at least in part byan FcRn gene. The FcRn may be from any organism, including but notlimited to humans, mice, rats, rabbits, and monkeys. As is known in theart, the functional FcRn protein comprises two polypeptides, oftenreferred to as the heavy chain and light chain. The light chain isbeta-2-microglobulin and the heavy chain is encoded by the FcRn gene.Unless other wise noted herein, FcRn or an FcRn protein refers to thecomplex of FoRn heavy chain with beta-2-microglobulin.

By “wildtype or parent polypeptide” as used herein is meant anunmodified polypeptide that is subsequently modified to generate avariant. The wildtype polypeptide may be a naturally occurringpolypeptide, or a variant or engineered version of a naturally occurringpolypeptide. Wildtype polypeptide may refer to the polypeptide itself,compositions that comprise the parent polypeptide, or the amino acidsequence that encodes it. Accordingly, by “wildtype immunoglobulin” asused herein is meant an unmodified immunoglobulin polypeptide that ismodified to generate a variant, and by “wildtype antibody” as usedherein is meant an unmodified antibody that is modified to generate avariant antibody. It should be noted that “wildtype antibody” includesknown commercial, recombinantly produced antibodies as outlined below.

The term “fragment crystallizable (Fc) polypeptide” is the portion of anantibody molecule that interacts with effector molecules and cells, Itcomprises the C-terminal portions of the immunoglobulin heavy chains.

The term “Framework” or “FR” refers to variable domain residues otherthan hypervariable region (HVR) residues. The FR of a variable domaingenerally consists of four FR domains: FR1, FR2, FR3, and FR4.Accordingly, the HVR and FR sequences generally appear in the followingsequence in VH (or VL): FR1-H1(L1) FR2-H2(L2) FR3-H3(L3)-FR4.

The terms “full length antibody,” “intact antibody.” and “wholeantibody” are used herein interchangeably to refer to an antibody havinga structure substantially similar to a native antibody structure orhaving heavy chains that contain an Fc region as defined herein.

A “functional Fc region” possesses an “effector function” of a nativesequence Fe region. Exemplary “effector functions” include C1q binding;complement dependent cytotoxicity; Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g. B cell receptor; BCR), etc.Such effector functions generally require the Fc region to be combinedwith a binding domain (e.g. an antibody variable domain) and can beassessed using various assays as herein disclosed, for example.

“Hinge region” is generally defined as stretching from Glu216 to Pro230of human IgG1 (Burton, Molec. Immunol. 22 (1985) 161-206). Hinge regionsof other IgG isotypes may be aligned with the IgGI sequence by placingthe first and last cysteine residues forming inter-heavy chain S═S bondsin the same positions.

The “lower hinge region” of an Fc region is normally defined as thestretch of residues immediately C-terminal to the hinge region, i.e.residues 233 to 239 of the Fc region.

“Homology” is defined as the percentage of residues in the amino acidsequence variant that are identical after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent homology.Methods and computer programs for the alignment are well known in theart. One such computer program is “Align 2”, authored by Genentech,Inc., which was filed with user documentation in the United StatesCopyright Office, Washington, D.C. 20559, on Dec. 10, 1991.

The terms “host cell,” “host cell line,” and “host cell culture” areused interchangeably and refer to cells into which exogenous nucleicacid has been introduced, including the progeny of such cells. Hostcells include “transformants” and “transformed cells,” which include theprimary transformed cell and progeny derived there from without regardto the nomber of passages. Progeny may not be completely identical innucleic acid content to a parent cell, but may contain mutations. Mutantprogeny that have the same function or biological activity as screenedor selected for in the originally transformed cell are included herein.

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human or a human cellor derived from a non-human source that utilizes human antibodyrepertoires or other buman. antibody-encoding sequences. This definitionof a human antibody specifically excludes a humanized antibodycomprising non-human antigen-binding residues.

“Human effector cells” are leukocytes which express one or more FcRs andperform effector functions, Preferably, the cells express at leastFcγRIII and perform ADCC effector function. Examples of human leukocyteswhich mediate ADCC include peripheral blood mononuclear cells (PBMC),natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils;with PBMCs and NK cells being preferred. The effector cells may beisolated from a native source thereof, e.g. from blood or PBMCs asdescribed herein.

A “humanized” antibody refers to a chimeric antibody comprising aminoacid. residues from non-human HVRs and amino acid residues from homanFRs. In certain embodiments, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the HVRs (e.g., CDRs) correspond tothose of a non-human antibody, and all or substantially all of the FRscorrespond to those of a human antibody. A humanized antibody optionallymay comprise at least a portion of an antibody constant region derivedfrom a human antibody. A “humanized form” of an antibody, e.g., anon-human antibody, refers to an antibody that has undergonehumanization.

The term “hypervariable region” or “HVR,” as used hercin, refers to eachof the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops (“'hypervariableloops”). Generally, native four-chain antibodies comprise six HVRs;three in the VH (H1, H2, H3), and three in the VL (L1, L2, L3). HVRsgenerally comprise amino acid residues from the hypervariable loopsand/or from. the “complementarity determining regions” (CDRs), thelatter being of highest sequence variability and/or involved in antigenrecognition. Exemplary hypervariable loops occur at amino acid residues26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101(H3) (Chothia, and Lesk. J. Mol. Biol. 196 (1987) 901-917). ExemplaryCDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) occur at aminoacid residues 24-34 of L1, 50-56 of L2, 89.97 of 13, 31-35B of H1, 50-65of H2, and 95-102 of H3 (Kabat, et al., Sequences of Proteins ofIumunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, MD (1991)). With the exception of CDRIin VH, CDRs generally comprise the amino acid residues that form thebypervariable loops. CDRs also comprise “specificity determiningresidues,” or “SDRs,” which are residues that contact antigen. SDRs arecontained within regions of the CDRs called abbreviated-CDRs, or a-CDRs.Exemplary a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, anda-CDR H3) occur at amino acid residues 31-34 of L1, 50-55 of L2, 89-96of L3, 31-35B of H1, 50-58 of H2, and 95-102 of H3 (See Almagro, andFransson, Front. Biosei. 13 (2008) 1619-1633). Unless otherwiseindicated, HVR residues and other residues in the variable domain (e.g.,FR residues) are numbered herein according to Kabat et al., supra.

“Immune complex” refers to the relatively stable structure which formswhen at least one target molecule and at least one heterologous Fcregion-containing polypeptide bind to one another forming a largermolecular weight complex. Examples of immune complexes areantigen-antibody aggregates and target molecule-immunoadhexinaggregates. The term “immune complex.” as used herein. unless indicatedotherwise, refers to an ex vivo complex (L.e. other than the form orsetting in which it may be found in nature). However, the immune complexmay be administered to a mammal, e.g. to evaluate clearance of theimmune complex in the mammal.

An “immunoconjugate” is an antibody conjugated to one or moreheterologous molecule(s), including but not limited to a cytotoxicagent.

An “individual” or “subject” is a mammal. Mammals include, but are notlimited to, domesticated animals (e.g., cows, sheep, cats, dogs, andhorses), primates (e.g., humans and non-human primates such as monkeys),rabbits, and rodents (e.g., mice and rats). In certain embodiments, theindividual or subject is a human.

An “isolated” antibody is one which has been separated from a componentof its natural environment. In some embodiments, an antibody is purifiedto greater than 95% or 99% purity as determined by, for example,electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillaryelectrophoresis) of chromatographic (e.g., ion exchange or reverse phaseHPLC). For review of methods for assessment of antibody purity, see,e.g., Flatman, et al., J. Chromatogr. B 848 (2007) 79-87.

An “isolated” polypeptide is one that has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials thatwould interfere with diagnostic or therapeutic uses for the polypeptide,and may include enzymes, hormones, and other proteinaceous ornonproteinaccous solutes. In preferred embodiments, the polypeptide willbe purified (1) to greater than 95% by weight of polypeptide asdetermined by the Lowry method, and most preferably more than 99% byweight, (2) to a degree sufficient to obtain at least 15 residues ofN-terminal or internal amino acid sequence by use of a spinning cupsequenator, or (3) to homogeneity by SDS-PAGE ander reducing ornonreducing conditions using Coomassie blue or, preferably, silverstain, Isolated polypeptide includes the polypeptide in situ withinrecombinant cells since at least one component of the polypeptide'snatural environment will not be present. Ordinarily, however, isolatedpolypeptide will be prepared by at least one purification step.

An “isolated” nucleic acid refers to a nucleic acid molecule that hasbeen separated from a component of its natural environment. An isolatednucleic acid includes a nucleic acid molecule contained in cells thatordinarily contain the nucleic acid molecule, but the nucleic acidmolecule is present extrachromosomally or at a chromosomal location thatis different from its natural chromosomal location.

“Isolated nucleic acid encoding an antibody refers to one or morenucleic acid molecules encoding antibody heavy and light chains (orfragments thereof), including such nucleic acid molecule(s) in a singlevector or separate vectors, and such nucleic acid molecule(s) present atone or more locations in a host cell.

The word “label” when used herein refers to a detectable compound orcomposition which is conjugated directly or indirectly to thepolypeptide. The label may be itself be detectable (e.g., radioisotopelabels or fluorescent labels) or, in the case of an enzymatic label, maycatalyze chemical alteration of a substrate compound or compositionwhich is detectable.

The term “ligand binding domain” as used herein refers to any nativecell-surface receptor or any region or derivative thereof retaining atleast a qualitative ligand binding ability of a corresponding nativereceptor. In a specific embodiment, the receptor is from a cell-surfacepolypeptide having an extracellular domain that is homologous to amember of the immunoglobulin supergenefamily. Other receptors, which arenot members of the immunoglobulin supergenefamily but are nonethelessspecifically covered by this definition, are receptors for cytokines,and in particular receptors with tyrosine kinase activity (receptortyrosine kinases) members of the hematopoietin and nerve growth factorreceptor superfamilies, and cell adhesion molecules, e.g. (E-, L- andP-) selectins.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variant antibodies,e.g., containing naturally occurring mutations or arising duringproduction of a monoclonal antibody preparation, such variants generallybeing present in minor amounts. In contrast to polyclonal antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody of amonoclonal antibody preparation is directed against a single determinanton an antigen. Thus, the modifier “monoclonal” indicates the characterof the antibody as being obtained from a substantially homogeneouspopulation of antibodies, and is not to be construed as requiringproduction of the antibody by any particular method. For example, themonoclonal antibodies to be used in accordance with the presentinvention may be made by a variety of techniques, including but notlimited to the hybridoma method, recombinant DNA methods, phage-displaymethods, and methods utilizing transgenic animals containing all or partof the human immunoglobulin loci, such methods and other exemplarymethods for making monoclonal antibodies being described herein.

A “naked antibody” refers to an antibody that is not conjugated to aheterologous moiety (e.g., a cytotoxic moiety) or radiolabel. The nakedantibody may be present in a pharmaceutical formulation.

“Native antibodies” refer to naturally occurring immunoglobulinmolecules with varying structures. For example, native IgG antibodiesare heterotetrameric glycoproteins of about 150,000 daltons, composed oftwo identical light chains and two identical heavy chains that aredisulfide-bonded. From N- to C-terminus, cach heavy chain has a variableregion (VH), also called a variable heavy domain or a heavy chainvariable domain, followed by three constant domains (CH1, CH2, and CH3).Similarly, from N- to C-terminus, each light chain has a variable region(VL), also called a variable light domain or a light chain variabledomain, followed by a constant light (CL) domain. The light chain of anantibody may be assigned to one of two types, called kappa (κ) andlambda (λ), based on the amino acid sequence of its constant andvariable dornain.

A “native sequence Fc region” comprises an amino acid sequence identicalto the amino acid sequence of an Fc region found in nature. Nativesequence human Fc regions inclode a native sequence human IgGI Fc region(non-A and A allotypes); native sequence human IgG2 Fc region; nativesequence human IgG3 Fc region; and native sequence human IgG4 Fc regionas well as naturally occurring variants thereof.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,combination therapy, contraindications and/or warnings concerning theuse of such therapeutic products.

By “position” as used herein is meant a location in the sequence of aprotein. Positions may be nombered sequentially, or according to anestablished format, for example the EU index for antibody numbering.

The terms “polypeptide” and “protein” are used interchangeably to referto a polymer of amino acid residues, comprising natural or non-naturalamino acid residues, and are not limited to a minimum length.

Thus, peptides, oligopeptides, dimers, multimers, and the like areincluded within the definition. Both full-length proteins and fragmentsthereof are encompassed by the definition. The terms also includepost-translational modifications of the polypeptide, including, forexample, glycosylation, sialylation, acetylation, and phosphorylation.

Furthermore, a “polypeptide” herein also refers to a modified proteinsuch as single ox multiple amino acid residue deletions, additions, andsubstitutions to the native sequence, as long as the protein maintains adesired activity. For example, a serine residue may be substituted toeliminate a single reactive cysteine or to remove disulfide bonding or aconservative amino acid substitution may be made to eliminate a cleavagesite. These modifications may be deliberate, as through site-directedmutagenesis, or may be accidental, such as through mutations of hostswhich produce the proteins or errors due to polymerase chain reaction(PCR) amplification.

The term “wildtype polypeptide” and “wildtype (human) Fc region” as usedherein refers to a polypeptide and Fc region, respectively, comprisingan amino acid sequence which lacks one or more of the Fc regionmodifications disclosed herein. because they have not been introduced,and serve for example as controls. The wildtype polypeptide may comprisea native sequence Fc region or an Fc region with pre-existing amino acidsequence modifications (such as additions, deletions and/orsubstitutions).

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of an activeingredient contained therein to be effective, and which contains noadditional componente which are unacceptably toxic to a subject to whichthe formulation would be administered.

A “pharmaceutically acceptable carrier” refers to an Ingredient in apharmaceutical formulation, other than an active ingredient, which isnontoxic to a subject. A pharmaceutically acceptable carrier includes,but is not limited to, a buffer, excipient, stabilizer, or preservative.

A polypeptide with “altered” FR binding affinity or ADCC activity is onewhich has either enhanced or diminished FcR binding activity and/or ADCCactivity compared to a parent polypeptide or to a polypeptide comprisinga native sequence Fc region. The polypeptide variant which “displaysincreased binding” to an FcR binds at least one FcR with better affinitythan the parent polypeptide. The polypeptide variant which “displaysdecreased binding” to an FcR, binds at least one FOR with worse affinitythan a parent polypeptide. Such variants which display decreased bindingto an FcR may possess little or no appreciable binding to an FcR, e.g.,0-20% binding to the FcR compared to a native sequence IgG Fc region,e.g. as determined in the Examples herein.

The polypeptide which binds an FcR with “reduced affinity” than a parentpolypeptide, is one which binds any one or more of the above identifiedFcRs with substantially reduced binding affinity than the parentantibody, when the amounts of polypeptide variant and parent polypeptidein the binding assay are essentially the same. For example, thepolypeptide variant with reduced FcR binding affinity may display fromabout 1.15 fold. to about 100 fold, e.g. from about 1.2 fold to about 50fold reduction in FcR binding affinity compared to the parentpolypeptide, where FcR binding affinity is determined, for example, asdisclosed in the Examples herein.

The polypeptide comprising an Fc variant which “mediatesantibody-dependent cell-mediated cytotoxicity (ADCC) in the presence ofhuman effector cells less effectively” than a parent or wildtypepolypeptide is one which in vitro or in vivo is substantially lesseffective at mediating ADCC, when the amounts of polypeptide variant andparent antibody used in the assay are essentially the same. Generally,such variants will be identified using the in vitro ADCC assay as hereindisclosed, but other assays or methods for determining ADCC activity,e.g. in an animal model etc, are contemplated. The preferred variant isfrom about 1.5 fold to about 100 fold, e.g. from about two fold to aboutfifty fold, less effective at mediating ADCC than the parent, e.g. inthe in vitro assay disclosed herein.

A “receptor” is a polypeptide capable of binding at least one ligand.The preferred receptor is a cell-surface receptor having anextracellular ligand-binding domain. and, optionally, other domains(e.g. transmembrane domain, intracellular domain and/or membraneanchor). The receptor to be evaluated in the assay described herein maybe an intact receptor or a fragment or derivative thereof (e.g. a fusionprotein comprising the binding domain of the receptor fused to one ormore heterologous polypeptides). Moreover, the receptor to be evaluatedfor its binding properties may be present in a cell or isolated andoptionally coated on an assay plate or some other solid phase.

The term “receptor binding domain” is used to designate any nativeligand for a receptor, including cell adhesion molecules, or any regionor derivative of such native ligand retaining at least a qualitativereceptor binding ability of a corresponding native ligand. Thisdefinition, among others, specifically includes binding sequences fromligands for the above-mentioned receptors.

As used herein, “treatment” (and grammatical variations thereof such as“treat” or “treating”) refers to clinical intervention in an attempt toalter the natural course of the individual being treated, and can beperformed either for prophylaxis or during the course of clinicalpathology. Desirable effects of treatment include, but are not limitedto, preventing occurrence or recurrence of disease, alleviation ofsymptoms, diminishment of any direct or indirect pathologicalconsequences of the disease, preventing metastasis, decreasing the rateof disease progression, amelioration or palliation of the disease state,and remission or improved prognosis. In some embodiments, antibodies ofthe invention are used to delay development of a disease or to slow theprogression of a disease.

By “variant protein” or “protein variant”, or “variant” as used hereinis meant a protein that differs from that of a parent protein by virtueof at least one amino acid modification, Protein variant may refer tothe protein itself, a composition comprising the protein, or the aminosequence that encodes it. Preferably, the protein variant has at leastone amino acid modification compared to the parent protein, e.g. fromabout one to about seventy amino acid modifications, and preferably fromabout one to about five amino acid modifications compared to the parent.The protein variant sequence herein will preferably possess at leastabout 80% homology with a parent protein sequence, and most preferablyat least about 90% homology, more preferably at least about 95%homology. Variant protein can refer to the variant protein itself,compositions comprising the protein variant, or the DNA sequence thatencodes it. Accordingly, by “antibody variant” or “variant antibody” asused herein is meant an antibody that differs from a parent antibody byvirtue of at least one amino acid modification, “IgG variant” or“variant IgG” as used herein is meant an antibody that differs from aparent IgG by virtue of at least one amino acid modification, and“immunoglobulin variant” or “variant immunoglobulin” as used herein ismeant an immunoglobulin sequence that differs from that of a parentimmunoglobulin sequence by virtue of at least one amino acidmodification.

The term “variable region” or “variable domain” refers to the domain ofan antibody heavy or light chain that is involved in binding theantibody to antigen. The variable domains of the heavy chain and lightchain (VH and VL, respectively) of a native antibody generally havesimilar structures, with each domain comprising four conserved frameworkregions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindt,et al., Kuby Immunology, 6th ed., W.H. Freeman and Co. (2007) page 91).A single VH or VL domain may be sufficient to confer antigen-bindingspecificity. Furthermore, antibodies that bind a particular antigen maybe isolated using a VH or VL domain from an antibody that binds theantigen to screen a library of complementary VL or VH domains,respectively. See, e.g., Portolano, et al., J. Immunol. 150 (1993)880-887; Clackson, et al., Nature 352 (1991) 624-628.

The term “vector,” as used herein, refers to a nucleic acid moleculecapable of propagating another nucleic acid to which it is linked, Theterm includes the vector as a self-replicating nucleic acid structure aswell as the vector incorporated into the genome of a host cell intowhich it has been introduced. Certain vectors are capable of directingthe expression of nucleic acids to which they are operatively linked.Such vectors are referred to herein as “expression vectors.”

The present application is directed to polypeptides that include aminoacid modifications that modalate binding to Fc receptors, inparticularly to Fcγ receptors.

DETAILED DESCRIPTION

The invention herein relates to a method for making a polypeptidecomprising a Fe variant. The “parent”, “starting” , “nonvariant” orwildtype polypeptide is prepared using techniques available in the artfor generating polypeptides or antibodies comprising an Fc region. Inthe preferred embodiment of the invention, the parent. polypeptide is anantibody and exemplary methods for generating antibodies are describedin more detail in the following sections. The parent polypeptide may,however, be any other polypeptide comprising an Fc region, e.g. animmunoadhesin. Methods for making immunoadhesins are elaborated in moredetail herein below.

In an alternative embodiment, a variant Fc region (Fc variant) may begenerated according to the methods herein disclosed and this Fc variantcan be fused to a heterologous polypeptide of choice, such as anantibody variable domain or binding domain of a receptor or ligand.

The wildtype polypeptide comprises an Fc region. Generally the Fc regionof the wildtype polypeptide will comprise a native or wildtype sequenceFc region, and preferably a human native sequence Fc region (human Fcregion). However, the Fc region of the wildtype polypeptide may have oneor more pre-existing amino acid sequence alterations or modificationsfrom a native sequence Fc region. For example, the C1q or Fcγ bindingactivity of the Fc region may have been previously altered (other typesof Fc region modifications are described in more detail below). In afurther embodiment the parent polypeptide Fc region is “conceptual” and,while it does not physically exist, the antibody engineer may decideupon a desired variant Fc region amino acid sequence and generate apolypeptide comprising that sequence or a DNA encoding the desiredvariant Fc region amino acid sequence.

In the preferred embodiment of the invention, however, a nucleic acidencoding an Fc region of a wildtype polypeptide is available and thisnucleic acid sequence is altered to generate a variant nucleic acidsequence encoding the Fc region variant.

DNA encoding an amino acid sequence variant of the starting polypeptideis prepared by a variety of methods known in the art. These methodsinclude, but are not limited to, preparation by site-directed (oroligonucleotide-mediated) mutagenesis, PCR mutagenesis, and cassettemutagenesis of an earlier prepared DNA encoding the polypeptide

Site-directed mutagenesis is a preferred method for preparingsubstitution variants. This technique is well known in the art (sec,e.g., Carter, et al., Nucleic Acids Res. 13 (1985) 4431-4443 and Kunkel,et al., Proc. Natl. Acad. Sci. USA 82 (1985) 488). Briefly, in carryingout site-directed mutagenesis of DNA, the starting DNA is altered byfirst hybridizing an oligonucleotide encoding the desired mutation to asingle strand of such starting DNA. After hybridization, a DNApolymerase is used to synthesize an entire second strand, using thehybridized oligonucleotide as a primer, and using the single strand ofthe starting DNA as a template. Thus, the oligonucleotide encoding thedesired mutation is incorporated in the resulting double stranded DNA.

PCR mutagenesis is also suitable for making amino acid sequence variantsof the starting polypeptide. See Higuchi, in PCR Protocols, AcademicPress (1990) pp. 177-183; and Vallette, et al., Nuc. Acids Res. 17(1989) 723-733. Briefly, when small amounts of template DNA are used asstarting material in a PCR, primers that differ slightly in sequencefrom the corresponding region in a template DNA can be used to generaterelatively large quantities of a specific DNA fragment that differs fromthe template sequence only at the positions where the primers differfrom the template.

Another method for preparing variants, cassette mutagenesis, is based onthe technique described by Wells, et al., Gene 34 (1985) 315-323.

One embodiment of the invention encompasses polypeptides comprising anFc region of an antibody, comprising the addition, substitution, ordeletion of at least one amino acid residue to the Fc region resultingin reduced or ablated affinity for at least one Fc receptor. The Fcregion interacts with a number of receptors or ligands including but notlimited to Fc Receptors (e.g., FcγRI, FcγRIIA, FcγRIIIA), the complementprotein C1q, and other molecules such as proteins A and G. Theseinteractions are essential for a variety of effector functions anddownstream signaling events including, but not limited to, antibodydependent cell-mediated cytotoxicity (ADCC), Antibody -dependentcellular phagocytosis (ADCP) and complement dependent cytotoxicity(CDC). Accordingly, in certain embodiments the variants of the inventionhave reduced or ablated affinity for an Fc receptor responsible for aneffector function compared to a polypeptide having the same amino acidsequence as the polypeptide comprising a Fc variant of the invention butnot comprising the addition, substitution, or deletion of at least oneamino acid residue to the Fc region (also referred to herein as an“wildtype polypeptide”). In certain embodiments, polypeptide comprisinga Fc variant of the invention comprise at least one or more of thefollowing properties: reduced or ablated effector (ADCC and/or CDCand/or ADCP) function, reduced or ablated binding to Fc receptors,reduced or ablated binding to C1q or reduced or ablated toxicities, Morespecifically, embodiments of the invention provide anti-CD20 (same asGA101 or GA), anti-CD9 (same as TA) and anti-Selectin (pSel) antibodieswith reduced affinity for Fo receptors (e.g. FcγRI, FcγRII, FcγRIIIA)and/or the complement protein C1q.

In one embodiment, antibodies of the invention comprise an Fc regioncomprising at least one addition, substitution, or deletion of an aminoacid residue at position P329, wherein the numbering system of theconstant region is that of the BU index as set forth in Kabat, et al.,NIH Publication 91 (1991) 3242, National Technical Information Service,Springfield, VA.

In a specific embodiment, polypeptides of the invention comprise an Fcvariant of a wild-type human Fc polypeptide said variant comprising anamino acid substitution at position Pro329, where the numbering of theresidues in the IgG Fc region is that of the EU index as in Kabat. Instill another embodiment, said variant comprises at least one furtheramino acid substitution.

In still another embodiment the polypeptide comprising a Fc variant of awild-type human Fc polypeptide has an amino acid substitution, deletionor addition which destroys or diminishes the function of the prolinesandwich in the region and/or interface of the Fc polypeptide with theFc Gamma receptor.

In another embodiment Pro329 is substituted with an amino acid which iseither smaller or larger then proline. In still another embodiment thesubstituted amino acid is Gly, Ala or Arg. In a further aspect of theinvention Pro329 of the Fc polypeptide is substituted with glycine,

In still another embodiment said polypeptide comprising a Fc variant hasat least one further amino acid substitution, addition of deletion. Instill another embodiment, said variants exhibit a reduced affinity to ahuman Fc receptor (FcγR) and/or a homan complement receptor as comparedto the polypeptide comprising the wildtype Fc polypeptide.

In another embodiment said polypeptide comprising a Fc variant exhibitsa reduced affinity to a human Pe receptor (FcγR) and/or a humancomplement receptor as compared to the polypeptide comprising thewildtype human Fc region, In a forther embodiment the affinity to atleast one of the FcγRI, FcγRII, FcγRIIIA is reduced, in a still furtherembodiment the affinity to the FcγRI and FcγRIIIA is reduced, and in astill further embodiment the affinity to the FcγRI, FcγRII and FcγRIIIAis reduced, in still a further aspect of the invention the affinity tothe FcγRI receptor, FcγRIIIA receptor and C1q is reduced, and in still afurther aspect of the invention the affinity to the FcγRI, FcγRII,FcγRIIIA and C1q receptor is reduced.

In still a further embodiment the ADCC induced by said polypeptidecomprising a Fc variant is reduced and in a preferred embodiment theADCC is reduced to al least 20% of the ADCC induced by the polypeptidecomprising the wildtype Fc polypeptide. In still a further aspect of theinvention, the ADCC and CDC induced by the polypeptide comprising thewildtype Fc polypeptide is decreased or ahlated and in a still furtheraspect the polypeptide comprising a Fc variant described above exhibit adecreased ADCC, CDC and ADCP compared to the polypeptide comprising thewildtype Fo polypeptide.

In one embodiment the at least one further amino acid substitution inthe polypeptide comprising the Fc variant is selected from the group:S228P, E233P, L234A, L235A, L235E, N297A, N297D, or P331S.

In a certain aspect of the invention the polypeptide comprising a Fcvariant comprises an antibody. In still another aspect of the inventionthe polypeptide comprising a Fc variant comprises a human IgG1 or IgG4Fc region. In still a further aspect of the invention the variants areIgG1 or IgG4 antibodies.

In another embodiment of the invention, polypeptides comprising a Pro329Fc variant variants further comprise at least one addition,substitution, or deletion of an amino acid residue in the Fc region thatis correlated with increased stability of the antibody. In still afurther aspect of the invention the affinity of the polypeptidecomprising a Fc variant described above to the Fon receptor is onlyslightly, and for example not more than 10-20% of the affinity ofpolypeptide comprising the wildtype Fc polypeptide altered.

In one embodiment, the addition, substitution, or deletion of an aminoacid residue in a polypeptide comprising a Fc variant is at position 228and/or 235 of the Fc region, wherein the numbering system of theconstant region is that of the EU index as set forth in Kabat, et al.

In a specific embodiment serine at position 228 and/or leucine atposition 235 in said polypeptide comprising a Fc variant is substitutedby another amino acid.

In a specific embodiment, polypeptides comprising a Fc variant of theinvention comprise an Fc region comprising an amino acid substitution atposition 225, wherein the serine residue is substituted with proline.

In a specific embodiment, polypeptides comprising a Fc variant of theinvention comprise an Fc region comprising an amino acid substitution atposition 235, wherein the leucine residue is substituted with glutamicacid.

In a specific embodiment the polypeptide comprising a Fc variantcomprises a triple mutation: an amino acid substitution at positionP329, a S228P and a L235E mutation (P329/SPLE).

In a further specific embodiment the polypeptide comprising a Fc variantcomprises a human IgG4 region.

In one embodiment, the addition, substitution, or deletion of an aminoacid residue is at position 234 and/or 235 of the Fc region, wherein thenumbering system of the constant region is that of the EU index as setforth in Kabat et al.

In a specific embodiment leucine at position 234 and/or leucine atposition 235 in the polypeptide comprising a Fc variant is substitutedby another amino acid.

In a specific embodiment, polypeptides comprising a Fc variant of theinvention comprise an Fc region comprising an amino acid substitution atposition 234, wherein the leucine residue is substituted with alanine.

In a specific embodiment, polypeptides comprising a Fc variant of theinvention comprise an Fc region comprising an amino acid substitution atposition 235, wherein the leucine residue is substituted with serine.

In a specific embodiment the polypeptide comprising an Fc variant of awildtype human Fc polypeptide comprises a triple mutation: an amino acidsubstitution at position Pro329, a L234A and a L235A mutation(P329/LALA).

In a further specific embodiment the above mentioned polypeptidescomprise a human IgGI region.

While it is preferred to alter binding to a FcγR, Fc region variantswith altered binding affinity for the neonatal receptor (FcRn) are alsocontemplated herein. Fc region variants with improved affinity for FcRnare anticipated to have longer serum half-lives, and such molecules willhave useful applications in methods of treating mammals where longhalf-life of the administered polypeptide is desired, e.g., to treat achronic disease or disorder. Fc region variants with decreased FcRnbinding affinity, on the contrary, are expected to have shorterhalf-lives, and such molecules may, for example, be administered to amammal where a shortened circulation time may be advantageous, e.g. forin vivo diagnostic imaging or for polypeptides which have toxic sideeffects when left circulating in the blood stream for extended periods,etc. Fc region variants with decreased FcRn binding affinity areanticipated to be less likely to cross the placenta, and thus may beutilized in the treatment of diseases or disorders in pregnant women.

Fc region variants with altered binding affinity for FoRn include thosecomprising an Fc region amino acid modification at any one or more ofamino acid positions 238, 252, 253, 254, 255. 256, 265, 272, 286, 288,303, 305, 307, 309, 311, 312, 317, 340, 356, 360, 362, 376, 378. 380,382, 386, 388, 400, 413, 415, 424, 433, 434, 435, 436, 439 or 447. Thosewhich display reduced binding to FcRn will generally comprise an Fcregion amino acid modification at any one or more of amino acidpositions 252, 253, 254, 255, 288, 309, 386, 388, 400, 415, 433, 435,436, 439 or 447; and those with increased binding to FcRn will usuallycomprise an Fc region amino acid modification at any one or more ofamino acid positions 238, 256, 265, 272, 286, 303, 305, 307, 311, 312,317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434.

In another embodiment, antibodies of the invention may be any of anyclass (for example, but not limited to IgG, IgM, and IgB). In certainembodiments, antibodies of the invention are members of the IgG class ofantibodies. In a specific embodiment, antibodies of the invention are ofthe IgG1, IgG2 or IgG4 subelass. In another specific embodiment,antibodies of the invention are of the IgGI subclass and comprise thefollowing amino acid substitutions: P329G and/or L234A and L235A of theFc region. In alternate embodiments, antibodies of the invention are ofthe IgG4 subclass, In a specific embodiment, antibodies of the inventionare of the IgG4 subclass and comprise the following amino acidsubstitutions: P329G and/or S228P and L235E of the Fc region. In certainembodiments, the modified antibodies of the present invention may beproduced by combining a variable domain, or fragment thereof, with an Fcdomain comprising one or more of the amino acid substitutions disclosedherein. In other embodiments modified antibodies of the invention may beproduced by modifying an Fc domain-containing antibody by introducingone or more of the amino acid substitutions residues into the Fc domain.

Reduced Binding to Fc Ligands

One skilled in the art will understand that antibodies of the inventionmay have altered (relative to an unmodified antibody) FcγR and/or C1qbinding properties (examples of binding properties include but are notlimited to, binding specificity, equilibrium dissociation constant(K_(D)), dissociation and association rates (kor and K_(on),respectively) binding affinity and/or avidity) and that certainalterations are more or less desirable. It is known in the art that theequilibrium dissociation constant (K_(D)) is defined as Kou/km. Oneskilled in the art can determine which kinetic parameter is mostimportant for a given antibody application. For example, a modificationthat reduces binding to one or more positive regulator (e.g., FcγRIIIA)and/or enhanced binding to an inhibitory Fc receptor (e.g., FcγRIIB)would be suitable for reducing ADCC activity. Accordingly, the ratio ofbinding affinities (e.g., equilibrium dissociation constants (K_(D)))can indicate if the ADCC activity of an antibody of the invention isenhanced or decreased. Additionally, a modification that reduces bindingto C1q would be suitable for reducing or eliminating CDC activity. Theaffinities and binding properties of an Fc region for its ligand, may bedetermined by a variety of in vitro assay methods (biochemical orimmunological based assays) known in the art for determining Fc-FcγR.interactions, i.e., specific binding of an Fc region to an FcγRincluding but not limited to, equilibrium methods (e.g., enzyme-linkedimmuno absorbent assay (ELISA) or radioimmunoassay (RIA)), or kinetics(c.g., BIACORE® analysis), and other methods such as indirect bindingassays, competitive inhibition assays, fluorescence resonance energytransfer (FRET), gel electrophoresis and chromatography (e.g., gelfiltration). These and other methods may utilize a label on one or moreof the components being examined and/or employ a variety of detectionmethods including but not limited to chromogenic, fluorescent,luminescent, or isotopic labels, A detailed description of bindingaffinities and kinetics can be found in Paul, W. E., ed., FundamentalImmunology, 4^(th) Ed., Lippincott-Raven, Philadelphia (1999).

In one aspect of the invention a polypeptide comprising an Fc variant ofa wild-type buman Fc region, said variant comprising an amino acidsubstitution at position Pro329 and at least one further amino acidsubstitution, exhibits a reduced affinity to a human Fc receptor (FcγR)and/or a human complement receptor as compared to the polypeptidecomprising the wildtype Fc polypeptide. In one aspect polypeptidescomprising an Fc variant of the invention exhibit affinities for a Fcreceptor that is at least 2 fold, or at least 3 fold, or at least 5fold, or at least 7 fold, or a least 10 fold, or at least 20 fold, or atleast 30 fold, or at least 40 fold, or at least 50 fold, or at least 60fold, or at least 70 fold, or at least 80 fold, or at least 90 fold, orat least 100 fold, or at least 200 fold less than for a wildtype Fcpolypeptide.

In one aspect polypeptides comprising an Fc variant of the inventionexhibit reduced binding affinity for one or more Fc receptors including,but not limited to FcγRI (CD64) including isoforms FcγRIA, FcγRII andFcγRIII (CD 16, including isoforms FcγRIIIA) as compared to anunmodified antibody.

In one aspect polypeptides comprising an Fc variant of the inventionexhibit reduced binding affinity for FcγRI (CD64) FcγRIIA and FcγRIIIAas compared to an unmodified antibody.

In one aspect polypeptides comprising an Fo variant of the inventionexhibit reduced binding affinity for FcγRIIA and FcγRIIIA as compared toan unmodified antibody.

In one aspect polypeptides comprising an Fc variant of the inventionexhibit reduced binding affinity for FcγRI (CD64) and FcγRIIIA ascompared to an unmodified antibody.

In one aspect of the invention polypeptides comprising an Fc variant ofthe invention exhibiting a reduced binding affinity for the Fc receptorsalso exhibit a reduced affinity to the C1q receptor.

In certain aspect polypeptides comprising an Fc variant of the inventiondo not comprise a concomitant increase in binding to the FcγRIIBreceptor as compared to a wildtype polypeptide. In certain aspects ofthe invention the polypeptides comprising an Fc variant have a reducedaffinity to the human receptor FcγIIIA, and to at least one furtherreceptor of the group comprising the human receptors FcγIIA, FcγIIIB,and C1q compared to the polypeptide comprising the wildtype Fepolypeptide. In further aspects of the invention polypeptides comprisingan Fe variant have a reduced affinity to the human receptor FcγIIIA, andto at two further receptors of the group comprising the human receptorsFcγIIA, FcγIIIB, and C1q compared to the polypeptide comprising thewildtype Fc polypeptide. In further aspect of the invention thepolypeptides comprising an Fc variant have a reduced affinity to thehuman FcγRIA, FcγIIIA, FcγIIA, FcγIIIB, and C1q compared to thepolypeptide comprising the wildtype Fc polypeptide. In still anotheraspect of the invention polypeptides comprising an Fc variant have areduced affinity to the human receptor FcγRIA, FcγIIIA, FcγIIA, FcγIIIB,and C1q compared to the polypeptide comprising the wildtype Fcpolypeptide.

In one aspect of the invention polypeptides comprising an Fc variant ofthe invention exhibit decreased affinities to FcγRI or FcγRIIA relativeto an unmodified antibody. In one aspect of the invention polypeptidescomprising an Fc variant exhibit affinities for FcγRI or FcγRIIA thatare at least 2 fold, or at least 3 fold, or at least 5 fold, or at least7 fold, or a least 10 fold, or at least 20 fold, or at least 30 fold, orat least 40 fold, or at least 50 fold, or at least 60 fold, or at least70 fold, or at least 80 fold, or at least 90 fold, or at least. 100fold, or at least 200 fold less than that of a wildtype polypeptide. Inone aspect of the invention polypeptides comprising an Fc variantexhibit affinity for the FcγRI or FcγRIIA that are at least 90%, atleast 809%, at least 70%, at least 60%, at least 50%, at least 40%, atleast 30%, at least 20%, at least 10%, or at least 5% less than a thanthat of a wildtype polypeptide.

In one aspect of the invention polypeptides comprising an Fc variant ofthe invention exhibit decreased affinity for the FcγRIIIA relative to anunmodified antibody. In one aspect polypeptides comprising an Fc variantof the invention exhibit affinities for FcγRIHIA that are at least 2foki, or at least 3 fold, or at least 5 fold, or at least 7 fold, or aleast 10 fold, or at least 20 fold, or at least 30 fold, or at least 40fold, or at least 50 fold, or at least 60 fold, or at least 70 fold, orat least 80 fold, or at least 90 fold, or at least 100 fold, or at least200 fold less than that of a wildtype polypeptide.

In one aspect of the invention polypeptides comprising an Fc variant ofthe invention exhibit affinities for FcγRIIIA that are at least 90%, atleast 80%, at least 70%, at least 60%, at least 50%, at least 40%, atleast 309%, at least 20%, at least 10%, or at least 5% less than that ofa wildtype polypeptide.

It is understood in the art that the F1-58V allelic variant of theFcγRIIIA has altered binding characteristics to antibodies. In oneembodiment, polypeptides comprising an Fc variant of the invention bindwith decreased affinities to FcγRIITA receptors relative to a wildtypepolypeptide. In one aspect polypeptides comprising an Fc variant of theinvention exhibit affinities for FcγRIIIA (F1 58V) that are at least 2fold, or at least 3 fold, or at least 5 fold, or at least 7 fold, or aleast 10 fold, or at least 20 fold, or at least 30 fold, or at least 40fold, or at least 50 fold, or at least 60 fold, or at least 70 fold, orat least 80 fold, or at least 90 fold, or at least 100 fold, or at least200 fold less than that of a wildtype polypeptide.

In one aspect of the invention polypeptides comprising an Fc variant ofthe invention exhibit decreased affinity for the C1q receptor relativeto an unmodified antibody. In one aspect polypeptides comprising an Fcvariant of the invention exhibit affinities for C1q receptor that are atleast 2 fold, or at least 3 fold, or at least 5 fold, or at least 7fold, or a least 10 fold, or at least 20 fold, or at least 30 fold, orat least 40 fold, or at least 50 fold, or at least 60 fold, or at least70 fold, or at least 80 fold, or at least 90 fold, or at least 100 fold,or at least 200 fold less than that of a wildtype polypeptide.

In one aspect of the invention polypeptides comprising an Fc variant ofthe invention exhibit affinities for C1q that are at least 90%, at least80%, at least 709%. at least 60%, at least 50%, at least 40%, at least30%, at least 20%, at least 10%, or at least 5% less than that of awildtype polypeptide.

In one aspect of the invention polypeptides comprising an Fc variant ofthe invention exhibit affinities for the human FcγRI, FcγRIIA, FcγRIIIA,FcγRIIIA (Fl 58V) or C1q receptors that are at least 90%, at least 80%,at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, atleast 20%, at least 10%, or at least 5% less than a wildtypepolypeptide.

In another aspect of the invention polypeptides comprising an Fc variantof the invention exhibit affinities for the FcγRI, FcγRIIA, FcγRIIIA,FcγRIHIA (Fl 58V) and/or C1q receptors, respectively, that are betweenabout 10 nM to 100 nM, 10 nM to 1 μM, 100 nM to about 100 μM, or about100 nM to about 10 μM, or about 100 nM to about 1 μM, or about 1 nM toabout 100 μM, or about 10 nM to about 100 μM, or about 1 μM to about 100μM, or about 10 μM to about 100 μM. In certain embodiments, polypeptidescomprising an Fc variant of the invention exhibit affinities for theFcγRI, FcγRIIA, FcγRIIIA, FcγRIIIA (FI-58V) or C1q receptors that aregreater than 100 nM, 500 nM, 1 μM, greater than 5 μM, greater than 10μM, greater than 25 μM, greater than 50 μM, or greater than 100 μM.

In another aspect of the invention polypeptides comprising an Fc variantof the invention exhibit increased affinities for the FcγRIIB ascompared to a wildtype polypeptide. In another aspect of polypeptidescomprising an Fc variant of the invention exhibit affinities for theFcγRHIB that are unchanged or increased by at least at least 2 fold, orat least 3 fold, or at least 5 fold, or at least 7 fold, or a least 10fold, or at least 20 fold, or at least 30 fold, or at least 40 fold, orat least SO fold, or at least 60 fold, or at least 70 fold, or at least80 fold, or at least 90 fold, or at least 100 fold, or at least 200 foldthan that of an unmodified antibody. In another aspect polypeptidescomprising an Fc variant of the invention exhibit affinities for theFcγRIIB receptor that are increased by at least 5%, at least 10%, atleast 20%, at least 30%, at least 405%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, or at least 95% than a wildtypepolypeptide.

In another aspect of the invention variants of the invention exhibitaffinities for the FcγRI, FcγRIIA FcγRIIIA, or FcγRIIIA (Fl 58V) or C1qreceptors that are less than 100 μM. less than 50 μM, less than 10 μM,less than 5 μM, less than 2.5 μM, less than 1 μM. or less than 100 nM,or less than 10 nM.

Reduced Effector Function

In a certain aspects of the invention polypeptides comprising an Fcvariant according to the invention modulate au effector function ascompared to the polypeptide comprising the wildtype Fc polypeptide.

In still another aspect of the invention this modulation is a modulationof ADCC and/or ADCP and/or CDC. In a further aspect of the inventionthis modulation is down-modulation or reduction in effect. In stillanother aspect of the invention this is a modulation of ADCC and stillin another aspect of the invention this modulation is a down-modulationof ADCC. In still another aspect this modulation is a down-modulation ofADCC and CDC, still in another embodiment this is a down-modulation isADCC only, in still another embodiment this is a down-modulation of ADCCand CDC and/or ADCP. In still another aspect of the invention thepolypeptides comprising an Fc variant according to the inventiondown-modulate or reduce ADCC/CDC and ADCP.

In a further aspect of the invention the reduction or down-modulation ofADCC of CDC or ADCP induced by the polypeptide comprising the Fcvariant, is a reduction to 0, 2.5, 5, 10, 20, 50 or 75% of the valueobserved for induction of ADCC, or CDC or ADCP, respectively, by thepolypeptide comprising the wildtype Fc region.

In still further aspects of the invention the modulation of ADCC inducedby the polypeptides comprising an Fc variant according to the inventionis a decrease in potency such that the ECSO of said Fc variant isapproximately >10-fold reduced compared to the polypeptide comprisingthe wildtype Fc polypeptide.

In still another aspect the variant according to the invention is devoidof any substantial ADCC and/or CDC and/or ADCP in the presence of humaneffector cells as compared to the polypeptide comprising the wildtype Fcpolypeptide.

In still another aspect of the invention the polypeptides comprising anFc variant of the invention exhibit a reduced, for example reduction byat least 20%, or strongly reduced, for example reduction by at least50%, effector function, which could be a reduction in ADCC(down-modulation), CDC and/or ADCP.

Reduced ADCC Activity

In vitro and/or in vivo cytotoxicity assays can be conducted to confirmthe reduction/depletion of CDC aud/or ADCC activities. For example, Pereceptor (FcR) binding assays can be conducted to ensure that theantibody lacks FcγR binding (hence likely lacking ADCC activity), butretains FcRn binding ability. The primary cells for mediating ADCC, NKcells, express FcγRILI only, whereas monocytes express FcγRI, RII andRIII. FOR expression on hematopoietic cells is summarized in Table 3 onpage 464 of Ravetch, and Kinet, Annu. Rev. Immunol. 9 (1991) 457-492.Non-limiting examples of in vitro assays to assess ADCC activity of amolecule of interest is described in U.S. Pat. No. 5,500,362 (see, e.g.Hellstrom, L, et al., Proc. Nat'l Acad. Sci. USA 83 (1986) 7059-7063)and Hellstrom, L, et al., Proc. Nat'l Acad. Sci. USA 82 (1985)1499-1502; U.S. Pat. No. 5,821,337 (see Bruggemann, M., et al., J. Exp.Med. 166 (1987) 1351-1361). Alternatively, non-radioactive assaysmethods may be employed (see, for example, ACTI™ non-radioactivecytotoxicity assay for flow cytometry (CellTechnology, Inc. MountainView, CA; and CytoTox 96® non-radioactive cytotoxicity assay (Promega,Madison, WI). Useful effector cells for such assays include peripheralblood mononuclear cells (PBMC) and Natural Killer (NK) cells.Alternatively, or additionally, ADCC activity of the molecule ofinterest may be assessed in vivo, e.g., in a animal model such as thatdisclosed in Clynes, et al., Proc. Nat'l Acad. Sci. USA 95 (1998)652-656. C1q binding assays may also be carried out to confirm that theantibody is unable to bind C1q and hence lacks CDC activity. See. e.g.,C1q and C3c binding ELISA in WO 2006/029879 and WO 2005/100402. Toassess complement activation, a CDC assay may he performed (see, forexample, Gazzano-Santoro, et al., J. Immunol. Methods 202 (1996) 163;Cragg, M. S., et al., Blood 101 (2003) 1045-1052; and Cragg, M. S., andGlennie, M. J., Blood 103 (2004) 2738-2743). FcRn binding and in vivoclearance/half life determinations can also be performed using methodsknown in the art (see, e.g., Petkova, S. B., et al., Int'l. Immunol.18(12) (2006) 1759-1769).

It is contemplated that polypeptides comprising a Fc variant of theinvention are characterized by in vitro functional assays fordetermining one or more FcγR mediated effector cell functions. Incertain embodiments, antibodies of the invention have similar bindingproperties and effector cell functions in in vivo models (such as thosedescribed and disclosed herein) as those in in vitro based assays.However, the present invention does not exclude variants of theinvention that do not exhibit the desired phenotype in in vitro basedassays but do exhibit the desired phenotype in vivo. In one embodiment,polypeptides comprising a Fc variant of the invention exhibit decreasedADCC activities as compared to an unmodified wildtype Fc polypeptides.In another aspect polypeptides comprising an Fc variant of the inventionexhibit ADCC activities that are at least 2 fold, or at least 3 fold, orat least S fold or at least 10 fold or at least 50 fold or at least 100fold less than that of an unmodified antibody. In still anotherembodiment, antibodies of the invention exhibit ADCC activities that arereduced by at least 10%, or at least 20%, or by at least 309h, or by atleast 40%, or by at least 50%, or by at least 60%, or by at least 70%,or by at least 80%, or by at least 90%, or by at least 100%, relative toan unmodified antibody. In a further aspect of the invention thereduction or down-modulation of ADCC induced by the polypeptidecomprising the Fc variant, is a reduction to 0, 2.5, 5, 10, 20, 50 or75% of the value observed for induction of ADCC, or CDC or ADCP,respectively, by the polypeptide comprising the wildtype Fc region. Incertain embodiments, polypeptides comprising an Fc variant of theinvention have no detectable ADCC activity. In specific embodiments, thereduction and/or ablation of ADCC activity may be attributed to thereduced affinity of the polypeptides comprising a Fc variant of theinvention for Fc ligands and/or receptors. In a specific embodiment ofthe invention the down-modulation of ADCC is a decrease in potency suchthat the EC50 of said polypeptide comprising an Fc variant isapproximately 10-fold of more reduced compared to the wildtype Fcpolypeptide.

In still another aspect the polypeptides comprising an Fc variantaccording to the invention modulate ADCC and/or CDC and/or ADCP. In aspecific aspect the variants according to the inventions show a reducedCDC and ADCC and/or ADCP activity.

Reduced CDC Activity

The complement activation pathway is initiated by the binding of thefirst component of the complement system (C1q) to a molecule, anantibody for example, complexed with a cognate antigen. To assesscomplement activation, a CDC assay. c.g, as described inGazzano-Santoro, et al, J. Immunol. Methods 202 (1996) 163, may beperformed.

The binding properties of the different variants to C1q can be analyzedby an ELISA sandwich type immunoassay. The antibody concentration at thehalf maximum response determines the ECse value. This read-out isreported as relative difference to the reference standard measured onthe same plate together with the coefficient of variation of sample andreference.

In one embodiment, polypeptides comprising an Fc variant according tothe invention exhibit decreased affinities to C1q relative to a wildtypepolypeptide. In another embodiment, of the polypeptides comprising a Fcvariant according to the invention exhibit affinities for C1q receptorthat are at least 2 fold, or at least 3 fold, or at least 5 fold, or atleast 7 fold, or a least 10 fold, or at least 20 fold, or at least 30fold, or at least 40 fold, or at least 50 fold, or at least 60 fold, orat least 70 fold, or at least 80 fold, or at least 90 fold, or at least100 fold, or at least 200 fold less than the wildtype polypeptide.

In another embodiment, polypeptides comprising a Pe variant according tothe invention exhibit affinities for C1q that are at least 90%, at least80%, at least 709%, at least 60%, at least 50%, at least 40%, at least30%, at least 20%, at least 10%, or at least 5% less than that of thewildtype polypeptide. In another embodiment, variants according to theinvention exhibit affinities for C1q that are between about 100 nM toabout 100 μM, or about 100 nM to about 10 μM, or about 100 nM to about 1μM, or about 1 nM to about 100 nM, or about 10 nM to about 100 μM, orabout 1 μM to about 100 μM, or about 10 μM to about 100 μM. In certainembodiments, polypeptides comprising an Fc variant of the inventionexhibit affinities for C1q that are greater than 1 μM, greater than 5μM, greater than 10 μM, greater than 25 μM, greater than 50 μM, orgreater than 100 μM.

In one embodiment polypeptide comprising an Fc variant of the inventionexhibit decreased CDC activities as compared to the wildtype Fcpolypeptide In another embodiment, polypeptide comprising an Fc variantof the invention exhibit CDC activities that are at least 2 fold, or atleast 3 fold, or at least 5 fold or at least 10 fold or at least 50 foldor at least 100 fold less than that of a wildtype polypeptide. In stillanother embodiment polypeptide comprising an Fc variant of the inventionexhibit CDC activities that are reduced by at least 10%, or at least20%, or by at least 30%, or by at least 40%, or by at least 50%, or byat least 60%, or by at least 70%, or by at least 80%, or by at least90%, or by at least 100%, or by at least 200%, or by at least 300%, orby at least 400%, or by at least 500% relative to the wildtypepolypeptide. In certain aspects polypeptide comprising an Fc variant ofthe invention exhibit no detectable CDC activities. In specificembodiments, the reduction and/or ablation of CDC activity may beattributed to the reduced affinity of the polypeptides comprising an Fcvariant for Fc ligands and/or receptors.

Reduced Antibody Related Toxicity

It is understood in the art that biological therapies may have adversetoxicity issues associated with the complex nature of directing theimmune system to recognize and attack unwanted cells and/or targets.When the recognition and/or the targeting for attack do not take placewhere the treatment is required, consequences such as adverse toxicitymay occur. For example, antibody staining of non-targeted tissues may beindicative of potential toxicity issues.

In one aspect, polypeptide comprising an Fc variant of the inventionexhibit reduced staining of non-targeted tissues as compared to thewildtype polypeptide. In another aspect, the polypeptide comprising anFc variant of the invention exhibit reduced staining of non-targetedtissues that are at least 2 fold, or at least 3 fold, or at least 5fold, or at least 7 fold, or a least 10 fold, or at least 20 fold, or atleast 30 fold, or at least 40 fold, or at least 50 fold, or at least 60fold, or at least 70 fold, or at least 80 fold, or at least 90 fold, orat least 100 fold, or at least 200 fold less than that of to a wildtypeFc polypeptide . In another embodiment, variants of the inventionexhibit reduced staining of non-targeted tissues that are reduced by atleast 10%, or at least 20%, or by at least 30%, or by at least 40%, orby at least 50%, or by at least 60%, or hy at least 70%, or by at least80%, or by at least 90%, or by at least 100%, or by at least 200%, or byat least 300%, or by at least 400%, or by at least 500% relative to thewildtype Fc polypeptide.

In one embodiment, polypeptides comprising an Fc variant of theinvention exhibit a reduced antibody related toxicity as compared to awildtype polypeptide, In another embodiment, polypeptide comprising anFc variant of the invention exhibit toxicities that are at least 2 fold,or at least 3 fold, or at least 5 fold, or at least 7 fold, or a least10 fold, or at least 20 fold, or at least 30 fold, or at least 40 fold,or at least 50 fold, or at least 60 fold, or at least 70 fold, or atleast 80 fold, or at least 90 fold, or at least 100 fold, or at least200 fold less than that of a wildtype polypeptide. In another aspect,polypeptides comprising an Fc variant of the invention exhibittoxicities that are reduced by at least 10%, or at least 20%, or by atleast 30%, or by at least 40%, or by at least 50%, or by at least 60%,or by at least 70%, or by at least 80%, or by at least 90%, or by atleast 100%, or by at least 200%, or by at least 300%, or by at least400%, or by at least 500% relative to the wildtype polypeptide.

Thrombocyte Aggregation

In one aspect of the invention the wildtype polypeptide induces plateletactivation and/or platelet aggregation, and the variants thereof, i.e.polypeptides, comprising Fc variants, show a decreased or even ablatedthrombocyte activation and/or aggregation. In still another aspect ofthe invention these wildtype polypeptides are antibodies targeting aplatelet protein. In yet another aspect the antibody is a CD9 antibody.In still another embodiment this CD9 antibody has a mutation at positionP329G and/or at position L234A/L235A or S228P/L235E (P329G/LALA,P329G/SPLE), In a further specific embodiment the antibody ischaracterized by the SEQ ID NOs: 8-14.

It is understood in the art that biological therapies may have asadverse effect thrombocyte aggregation. In vitro and in vivo assayscould be used for measuring thrombocyte aggregation. It is assumed thatthe in vitro assay reflects the in vivo situation.

In one aspect, polypeptides comprising an Fc variant of the inventionexhibit reduced thrombocyte aggregation in an in vitro assay compared tothe wildtype polypeptide. In another aspect, polypeptides comprising anFc variant of the invention exhibit reduced thrombocyte aggregation inan in vitro assay that is at least 2 fold, or at least 3 fold, or atleast 5 fold, or at least 7 fold, or a least 10 fold, or at least 20fold, or at least 30 fold, or at least 40 fold, or at least 50 fold, orat least 60 fold, or at least 70 fold, or at least 80 fold, or at least90 fold, or at least 100 fold, or at least 200 fold less than that ofthe wildtype polypeptide. In another embodiment, polypeptides comprisingan Fc variant of the invention exhibit reduced thrombocyte aggregationin an in vitro assay that is reduced by at least 10%, or at least 20%,or by at least 30%, or by at least 40%, or by at least 50%, or by atleast 60%, or by at least 70%, or by at least 80%, or by at least 90%,or by at least 100%, or by at least 200%, or by at least 300%, or by atleast 400%, or by at least 500% relative to the wildtype polypeptide.

In still another aspect, polypeptides comprising an Pe variant of theinventions exhibit a reduced in vivo thrombocyte aggregation compared tothe wildtype polypeptide. In another aspect, variants of the inventionexhibit reduced thrombocyto aggregation in an in vivo assay that is atleast 2 fold, or at least 3 fold, or at least 5 fold, or at least 7fold, or a least 10 fold, or at least 20 fold, or at least 30 fold, orat least 40 fold, or at least 50 fold, or at least 60 fold, or at least70 fold, or at least 80 fold, or at least 90 fold, or at least 100 fold,or at least 200 fold less than that of the wildtype Fc polypeptide. Inanother embodiment, polypeptides comprising an Fc variant of theinvention exhibit reduced thrombocyte aggregation in an in vivo assaythat is reduced by at least 10%, or at least 20%, or by at least 30%, orby at least 40%, or by at least 509%, or by at least 60%, or by at least70%, or by at least 80%, or by at least 90%, or by at least 100%, or byat least 200%, or by at least 300%, or by at least 400%, or by at least500% relative to the wildtype polypeptide.

Internalizing Antibodies

Variants of the invention may bind to cell-surface antigens that mayinternalize, further carrying the antibodies into the cell. Once insidethe cell, the variants may be released into the cytoplasm, targeted to aspecific compartment, or recycled to the cell surface. In someembodiments, the variants of the invention bind to a cell-surfaceantigen that internalizes. In other embodiments, antibodies of theinvention may be targeted to specific organelles or compartments of thecell. In yet other embodiments, the variants of the invention may berecycled to the cell surface or periphery after internalization.

In a specific embodiment, the antibody of the invention is specific forp-Selectin, CD9, CD19, CD81, CCRS or CXCR5, IL17a or Il-33.

Antibody Preparation

In the preferred embodiment of the invention, the Fc region-containingpolypeptide which is modified according to the teachings herein is anantibody. Techniques for producing antibodies follow:

Antigen Selection and Preparation

Where the polypeptide is an antibody, it is directed against an antigenof interest. Preferably, the antigen is a biologically importantpolypeptide and administration of the antibody to a mammal sufferingfrom a disease or disorder can result in a therapeutic benefit in thatmammal. However, antibodies directed against nonpolypeptide antigens(such as tumor-associated glycolipid antigens; see U.S. Pat. No.5,001,178) are also contemplated.

Where the antigen is a polypeptide, it may be a transmembrane molecule(e.g. receptor) or ligand such as a growth factor. Exemplary antigensinclude molecules such as renin; a growth hormone, including humangrowth hormone and bovine growth hormone; growth hormone releasingfactor; parathyroid hormone; thyroid stimulating hormone; lipoproteins;alpha-1-antitrypsin; insulin A-chain; insulin B-chain; proinsulin;follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon;clotting factors such as factor VIIIC, factor IX, tissue factor (TF),and von Willebrands factor; anti-clotting factors such as Protein C;atrial natriuretic factor; lung surfactant; a plasminogen activator,such as urokinase or human urine or tissne-type plasminogen activator(t-PA): bombesin; thrombin; bemopoietic growth factor; tumor necrosisfactor-alpha and -beta; enkephalinase; RANTES (regulated on activationnormally T-cell expressed and secreted); human macrophage inflammatoryprotein (MIP-1-alpha); a serum albumin such as human serum albumin;Muellerian-inhibiting substance; relaxin A-chain; relaxin B-chain;prorelaxin; mouse gonadotropin-associated peptide; a microbial protein,such as beta-lactamase: DNase; IgE; a cytotoxic T-lymphocyte associatedantigen (CTLA), such as CTLA-4; inhibin; activin; vascular endothelialgrowth factor (VEGF); receptors for hormones or growth factors; proteinA or D; rheumatoid factors; a neurotrophic factor such as bone-derivedneurotrophic factor (BDNF), neurotrophin-3, 4, 5, or -6 (NT-3, NT-4,NT-5, or NT-6), or a nerve growth factor such as NGF.B; platelet-derivedgrowth factor (PDGF); fibroblast growth factor such as aFGF and bFGF;epidermal growth factor (EGF); transforming growth factor (TGF) such asTGF-alpha and TGF-beta, including TOF-B1, TGF-B2, TGF-B3, TGF-B4, orTGF-β5; insulin-like growth factor-I and -II (IGF-I and IGF-II);des(1-3)-IGF-I (brain IGF-I), insulin-like growth factor bindingproteins; CD proteins such as CD4, CDS, CD19 and CD20; erythropoietin;osteoinductive factors; immunotoxins; a bone morphogenetic protein(BMP); an interferon such as interferon-alpha, -beta, and -gamma; colonystimulating factors (CSFs), e.g., M-CSE, GM-CSF, and G-CSF; interleukins(ILs), e.g., IL-1 to IL-10; superoxide dismutase; surface membraneproteins; decay accelerating factor; viral antigen such as, for example,a portion of the AIDS envelope; transport proteins; homing 30 receptors;addressins; regulatory proteins; integrins such as CD11a, CD11b, CD11c,CD18, an ICAM, VLA-4 and VCAM; a tumor associated antigen such as HER2,HER3 or HER4 receptor; and fragments of any of the above-listedpolypeptides.

Preferred molecular targets for antibodies encompassed by the presentinvention include CD proteins such as CD4, CD8, CD19, CD20 and CD34;members of the ErbB receptor family such as the BGF receptor, HER2, HER3or HER4 receptor; cell adhesion molecules such as LFA-1, Mac1, p150.95,VLA-4, ICAM-1, VCAM, α4/β7 integrin, and αv/β3 integrin including eitherα or β subunits thereof (e.g. anti-CDIIa, anti-CD18 or anti-CD11bantibodies); growth factors such as VEGF; tissue factor (TF); alphainterferon (α-IFN); an interleukin, such as IL-8; IgB; blood groupantigens; flk2/flt3 receptor; obesity (OB) receptor; mp1 receptor;CTLA-4; protein C etc.

Soluble antigens or fragments thereof, optionally conjugated to othermolecules, can be used as immunogens for generating antibodies. Fortransmembrane molecules, such as receptors, fragments of these (e.g. theextracellular domain of a receptor) can be used as the immunogen.Alternatively, cells expressing the transmembrane molecule can be usedas the immunogen. Such cells can be derived from a natural source (e.g.cancer cell lines) or may be cells which have been transformed byrecombinant techniques to express the transmembrane molecule. Otherantigens and forms thereof useful for preparing antibodies will beapparent to those in the art.

Polyclonal Antibodies

Polyclonal antibodies are preferably raised in animals by multiplesubcutaneous (sc) or intraperitoneal (ip) injections of the relevantantigen and an adjuvant. It may be useful to conjugate the relevantantigen to a protein that is immunogenic in the species to be immunized,e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, orsoybean trypsin inhibitor asing a bifunctional or derivatizing agent,for example, maleimidobenzoyl sulfosuccinimide ester (conjugationthrough cysteine residues), N-hydroxysuccinimide (through lysineresidues), glutaraldehyde, succinic anhydride, SOCH, or carbodiimidewhere R and R¹ are different alkyl groups.

Animals are immunized against the antigen, immunogenic conjugates, orderivatives by combining, c.g., 100 μg or 5 μg of the protein orconjugate (for rabbits or mice, respectively) with 3 volumes of Freund'scomplete adjuvant and injecting the solution intradermally at multiplesites. One month later the animals are boosted with for example 1/10 ofthe original amount of peptide or conjugate in Freund's completeadjuvant by subcutaneous injection at multiple sites. Seven to 14 dayslater the animals are bled and the serum is assayed for antibody titer.Animals are boosted until the titer plateaus. Preferably, the animal isboosted with the conjugate of the same antigen, but conjugated to adifferent protein and/or through a different cross-linking reagent.Conjugates also can be made in recombinant cell culture as proteinfusions. Also, aggregating agents such as alum are suitably used toenhance the immune response.

Monoclonal Antibodies

Monoclonal antibodies may be made using the hybridoma method firstdescribed. by Kohler, et al., Nature, 256 (1975) 495, or may be made byrecombinant DNA methods (U.S. Pat. No. 4,816,567).

In the hybridoma method, a mouse or other appropriate host animal, suchas a hamster or macaque monkey, is immunized as hereinabove described toelicit lymphocytes that produce or are capable of producing antibodiesthat will specifically bind to the protein used for immunization.Alternatively, lymphocytes may be immunized in vitro. Lymphocytes thenare fused with myeloma cells using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, Academic Press (1986) pp. 59-103).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stablehigh-level production of antibody by the selected antibody-producingcells, and are sensitive to a medium such as HAT medium. Among these,preferred myeloma cell lines are murine myeloma lines, such as thosederived from MOPC-21 and MPC-11 mouse tumore available from the SalkInstitute Cell Distribution Center, San Diego, Calif. USA, and SP-2 orX63-Ag8-653 cells available from the American Type Culture Collection,Rockville, Md. USA. Human myeloma and mouse-human heteromyeloma celllines also have been described for the production of human monoclonalantibodies (Kozbor, J., Inmunol. 133 (1984) 3001; Brodeur, et al.,Monoclonal Antibody Production Techniques and Applications, MarcelDekker, Inc., New York (1987) pp. 51-63).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the antigen.Preferably, the binding specificity of monoclonal antibodies produced bybybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besuhcloned by limiting dilation procedures and grown by standard methods(Goding, Monoclonal Antibodies: Principles and Practice, Academic Press(1986) pp. 59-103). Suitable culture media for this purpose include, forexample, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells maybe grown in vivo as ascites tumors in an animal,

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

DNA encoding the monoclonal antibodies is readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of the monoclonal antibodies). The hybridoma cells serve asa preferred source of such DNA. Once isolated, the DNA may be placedinto expression vectors, which are then transfected into host cells suchas E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells,or myeloma cells that do not otherwise produce immunoglobulin protein,to obtain the synthesis of monoclonal antibodies in the recombinant hostcells. Recombinant production of antibodies will be described in moredetail below.

In a further embodiment, antibodies or antibody fragments can beisolated from antibody phage libraries generated using the techniquesdescribed in McCafferty, J., et al., Nature 348 (1990) 552-554.Clackson, et al., Nature 352 (1991) 624-628 and Marks, et al., J. Mol.Biol. 222 (1991) 581-597 describe the isolation of murine and humanantibodies, respectively, asing phage libraries. Subsequent publicationsdescribe the production of high affinity (nM range) human antibodies bychain shuffling (Marks, et al., Bio/Technology 10 (1992) 779-783), aswell as combinatorial infection and in vivo recombination as a strategyfor constructing very large phage libraries (Waterhouse, et al., Noc.Acids. Res. 21 (1993) 2265-2266). Thus, these techniques are viablealternatives to traditional monoclonal antibody hybridoma techniques forisolation of monoclonal antibodies.

The DNA also may be modified, for example, by substituting the codingsequence for human heavy- and light-chain constant domains in place ofthe homologous murine sequences (U.S. Pat. No. 4,816,567; Morrison, etal., Proc. Natl. Acad. Sci. USA, 81 (1984) 6851-6855), or by covalentlyjoining to the immunoglobulin coding sequence all or part of the codingsequence for a non-immunoglobulin polypeptide.

Typically such non-immunoglobulin polypeptides are substituted for theconstant domains of an antibody, or they are substituted for thevariable domains of one antigen combining site of an antibody to createa chimeric bivalent antibody comprising one antigen-combining sitehaving specificity for an antigen and another antigen-combining sitehaving specificity for a different antigen.

Antibody Affinity

In certain embodiments, an antibody provided herein has a dissociationconstant (Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or≤0.001 nM (e.g. 10⁻⁸ M or less, e.g. from 10⁻⁸ M to 10⁻¹³ M, e.g., from10⁻⁹ M to 10⁻¹³ M).

In one embodiment, Kd is measured by a radiolabeled antigen or Fcreceptor binding assay (RIA) performed with the Fab version of anantibody of interest and its antigen as described by the followingassay. Solution binding affinity of Fabs for antigen is measured byequilibrating Fab with a minimal concentration of (¹²⁵I)-labeled antigenin the presence of a titration series of unlabeled antigen, thencapturing bound antigen with an anti-Fab antibody-coated plate (see,e.g., Chen, et al., J. Mol. Biol. 293 (1999) 865-881). To establishconditions for the assay. MICROTITER® multi-well plates (ThermoScientific) are coated overnight with 5 ng/ml of a capturing anti-Fabantibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6). andsubsequently blocked with 2% (w/v) bovine serum albumin in PBS for twoto five hours at room temperature (approximately 23° C.). In anon-adsorbent plate (Nunc #269620), 100 μM or 26 pm [¹²⁵I]-antigen aremixed with serial dilutions of a Fab of interest (e.g., consistent withassessment of the anti-VEGF antibody, Fab-12, in Presta, et al., CancerRes. 57 (1997) 4593-4599). The Fab of interest is then incubatedovernight; however, the incubation may continue for a longer period(c.g., about 65 hours) to ensure that equilibrium is reached.

Thereafter, the mixtures are transferred to the capture plate forincubation at room temperature (e.g., for one hour). The solution isthen removed and the plate washed eight times with 0.1% polysorbate 20(TWEEN-20®) in PBS. When the plates have dried, 150 μl/well ofscintillant (MICROSCINT-20™; Packard) is added, and the plates arecounted on a TOPCOUNT™ gamma counter (Packard) for ten minutes.Concentrations of each Fab that give less than or equal to 20% ofmaximal binding are chosen for use in competitive binding assays.

According to another embodiment, Kd is measured using surface plasmonresonance assays using a BIACORE®-2000 or a BIACORE 2-3000 (BIAcore,Inc., Piscataway, NJ) at 25° C. with immobilized antigen or Fc receptorCM5 chips at ˜10 response units (RU). Briefly, carboxymethylated dextranbiosensor chips (CM5, BIACORE, Inc.) are activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions.Antigen is diluted with 10 mM sodium acetate, pH 4.8. to 5 μg/ml (˜0.2μM) before injection at a flow rate of 5 μl/minute to achieveapproximately 10 response units (RU) of coupled protein, Following theinjection of antigen, 1 M ethanolamine is injected to block anreactedgroups. For kinetics measurements, two-fold serial dilutions of Fab(0.78 nM to 500 nM) are injected in PBS with 0.05% polysorbate 20(TWEEN-201™) surfactant (PBST) at 25° ° C. at a flow rate ofapproximately 25 μl/min. Association rates (k_(on)) and dissociationrates (k_(off)) are calculated using a simple one-to-one Langmuirbinding model (BIACORE Evaluation Software version 3.2) bysimultaneously fitting the association and dissociation sensograms. Theequilibrium dissociation constant (Kd) is calculated as the ratiok_(off)/k_(on). See, e.g., Chen, et al., J. Mol. Biol. 293 (1999)865-881. If the on-rate exceeds 10⁶ M⁻¹s⁻¹ by the surface plasmon 25resonance assay above, then the on-rate can be determined by using afluorescent quenching technique that measures the increase or decreasein fluorescence emission intensity (excitation=295 nm; emission=340 nm,16 nm band-pass) at 25° C. of a 20 nM anti-antigen antibody (Fab form)in PBS, pH 7.2, in the presence 30 of increasing concentrations ofantigen as measured in a spectrometer, such as a stop-flow equippedspectrophometer (Aviv Instruments) or a 8000-series SLM-AMINCO™spectrophotometer (ThermoSpectronic) with a stirred cuvette.

In certain embodiments, an antibody provided herein is an antibodyfragment. Antibody fragments include, but are not limited to, Fab, Fab′,Fab′-SH, F(ab′)₂, Fv, and scFv fragments, and other fragments describedbelow. For a review of certain antibody fragments, see Hudson, et al.,Nat. Med. 9 (2003) 129-134. For a review of scFv fragments, see, e.g.,Pluekthün, in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315(1994); see also WO 93/16185; and U.S. Pat. Nos. 5,571,894 and5,587,458. For discussion of Fab and F(ab′); fragments comprisingsalvage receptor binding epitope residues and having increased in vivohalf-life, see U.S. Patent No. US 5,869,046.

Diabodies are antibody fragments with two antigen-binding sites that maybe bivalent or bispecific. See, for example, EP 0 404 097; WO1993/01161; Hudson, et al., Nat. Med. 9 (2003) 129-134; and Hollinger,et al., Proc. Natl. Acad. Sci. USA 90 (1993) 6444-6448. Triabodies andtetrabodies are also described in Hudson, et al., Nat. Med. 9 (2003)129-134.

Single-domain antibodies are antibody fragments comprising all or aportion of the heavy chain variable domain or all or a portion of thelight chain variable domain of an antibody. In certain embodiments, asingle-domain antibody is a human single-domain antibody (Domantis,Inc., Waltham, MA; see, e.g., U.S. Pat. No. 6,248,516 B1).

Antibody fragments can be made by various techniques, including but notlimited to proteolytic digestion of an intact antibody as well asproduction by recombinant host cells (e.g. E. coli or phage), asdescribed herein.

Chimeric and Humanized Antibodies

In certain embodiments, an antibody provided herein is a chimericantibody. Certain chimeric antibodies are described, e.g., in U.S. Pat.No. 4,816,567; and Morrison, et al., Proc. Natl. Acad. Sci. USA, 81(1984) 6851-6855). In one example, a chimcric antibody comprises anon-human variable region (e.g., a variable region derived from a mouse,rat, hamster, rabbit, or non-human primate, such as a monkey) and ahuman constant region. In a further example, a chimeric antibody is a“class switched” antibody in which the class or subclass has beenchanged from that of the parent antibody. Chimeric antibodies includeantigen-binding fragments thereof.

In certain embodiments, a chimerie antibody is a humanized antibody.Typically, a non-human antibody is humanized to reduce immunogenicity tohumans, while retaining the specificity and affinity of the parentalnon-human antibody. Generally, a humanized antibody comprises one ormore variable domains in which HVRs, e.g., CDRs, (or portions thereof)are derived from a non-human antibody. and FRs (or portions thereof) arederived from human antibody sequences. A. humanized antibody optionallywill also comprise at least a portion of a human constant region. Insome embodiments, some FR residues in a humanized antibody aresubstituted with corresponding residues from a non-human antibody (e.g.,the antibody from which the HVR residues are derived), e.g., to restoreor improve antibody specificity or affinity.

Humanized antibodies and methods of making them are reviewed, e.g., inAlmagro, and Fransson, Front. Biosci. 13 (2008) 1619-1633, and arefurther described, e.g., in Riechmann, et al., Nature 332 (1988)323-329; Queen, et al., Proc. Nat'l Acad. Sci. USA 86 (1989)10029-10033; U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and7,087,409; Kashmiri, et al., Methods 36 (2005) 25-34 (describing SDR(a-CDR) grafting); Padlan, Mol. Immunol. 28 (1991) 489-498 (describing“resurfacing”); Dall'Acqua, et al., Methods 36 (2005) 43-60 (describing“FR shuffling”); and Osboum, et al., Methods 36 (2005)61-68 and Klimka,et al., Br. J. Cancer, 83 (2000) 252-260 (describing the “guidedselection” approach to FR shuffling).

Human framework regions that may be used for humanization include butare not limited to: framework regions selected using the “best-fit”method (see, e.g., Sims, et al., J. Immunol. 151 (1993) 2296); frameworkregions derived from the consensus sequence of human antibodies of aparticular subgroup of light or heavy chain variable regions (see, e.g.,Carter, et al., Proc. Natl. Acad. Sci. USA, 89 (1992) 4285; and Presta,et al., J. Immunol., 151 (1993) 2623); human mature (somaticallymutated) framework regions or human germline framework regions (see,e.g., Almagro, and Fransson, Front. Biosci. 13 (2008) 1619-1633); andframework regions derived from screening FR libraries (sce, c.g., Baca,et al., J. Biol. Chem. 272 (1997) 10678-10684 and Rosok, et al., J.Biol. Chem. 271 (1996) 22611-22618).

Human Antibodies

In certain embodiments, an antibody provided herein is a human antibody.Haman antibodies can be produced using various techniques known in theart. Human antibodies are described generally in van Dijk, and van deWinkel, Curr. Opin. Pharmacol. 5 (2001) 368-74 and Lonberg, Carr. Opin.Immunol. 20 (2008) 450-459.

Human antibodies may be prepared by administering an immunogen to atransgenic animal that has been modified to produce intact humanantibodies or intact antibodies with human variable regions in responseto antigenic challenge. Such animals typically contain all or a portionof the human immunoglobulin loci, which replace the endogenousimmunoglobulin loch, or which are present extrachromosomally orintegrated randomly into the animal's chromosomes. In such transgenicmice, the endogenous immunoglobulin loci have generally beeninactivated. For review of methods for obtaining human antibodies fromtransgenic animals, see Lonberg, Nat. Biotech. 23 (2005) 1117-1125. Seealso, c.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 describing XENOMOUSE™technology; U.S. Pat. No. 5,770,429 describing HUMABO technology; U.S.Pat. No. 7,041,870 describing K-M MOUSE® technology, and U.S. PatentApplication Publication No. US 2007/0061900, describing VELOCIMOUSE®technology). Human variable regions from intact antibodies generated bysuch animals may be further modified, e.g., by combining with adifferent human constant region.

Human antibodies can also be made by hybridoma-based methods. Humanmyeloma and mouse-human beteromyeloma cell lines for the production ofhuman monoclonal antibodies have been described. (See, e.g., Kozbor, J.Immunol., 133 (1984) 3001; Brodeur, et al., Monoclonal AntibodyProduction Techniques and Applications, Marcel Dekker, Inc., New York,(1987) pp. 51-63: and Boerner, et al., J. Immunol., 147 (1991) 86.)Human antibodies generated via human B-cell hybridoma technology arealso described in Li, et al., Proc. Natl. Acad. Sci. USA, 103 (2006)3557-3562. Additional methods include those described, for example, inU.S. Pat. No. 7,189,826 (describing production of monoclonal human IgMantibodies from hybridoma cell lines) and Ni, Xiandal Mianyixue, 26(4)(2006) 265-268 (describing human-human hybridomas), Human hybridomatechnology (Trioma technology) is also described in Vollmers, andBrandlein, Histology and Flistopathology 20(3) (2005) 927-937 andVollmers, and Brandlein, Methods and Findings in Experimental andClinical Pharmacology 27(3) (2005) 185-91.

Human antibodies may also be generated by isolating Fv clone variabledomain sequences selected from human-derived phage display libraries.Such variable domain sequences may then be combined with a desired humanconstant domain. Techniques for selecting homan antibodies from antibodylibraries are described below.

Library-Derived Antibodies

Antibodies of the invention may be isolated by screening combinatoriallibraries for antibodies with the desired activity or activities. Forexample, a variety of methods are known in the art for generating phagedisplay libraries and screening such libraries for antibodies possessingthe desired binding characteristics. Such methods are reviewed, e.g., inHoogenboom, H. R., et al., in Methods in Molecular Biology 178 (2002)1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001) and furtherdescribed, e.g., in the McCafferty, J., et al., Nature 348 (1990)552-554; Clackson, et al., Nature 352 (1991) 624-628; Marks, et al., J.Mol. Biol. 222 (1992) 581-597; Marks, and Bradbury, in Methods inMolecular Biology 248 161-175 (Lo, ed., Human Press, Totowa, NJ, 2003);Sidhu, et al., J. Mol. Biol. 338(2) (2004) 299-310; Lee, et al., J. Mol.Biol. 340(5) (2004) 1073-1093; Fellouse, Proc. Natl. Acad. Sci. USA101(34) (2004) 12467-12472; and Lee, et al., J. Immunol. Methods284(1-2) (2004) 119-132.

In certain phage display methods, repertoires of VII and VL genes areseparately cloned by polymerase chain reaction (PCR) and recombinedrandomly in phage libraries, which can then be screened forantigen-binding phage as described in Winter, et al., Ann. Rev.Immunol., 12 (1994) 433-455. Phage typically display antibody fragments,either as single-chain Fv (scFv) fragments or as Fab fragments.Libraries from immunized sources provide high-affinity antibodies to theimmunogen without the requirement of constructing hybridomas,Alternatively, the naive repertoire can be cloned (e.g., from human) toprovide a single source of antibodies to a wide range of non-self andalso self antigens without any immunization as described by Griffiths,et al., EMBO J, 12 (1993) 725-734. Finally, naive libraries can also bemade synthetically by cloning unrearranged V-gene segments from stemcells. and using PCR primers containing random sequence to encode thehighly variable CDR3 regions and to accomplish rearrangement in vitro,as described by Hoogenboom, and Winter, J. Mol. Biol., 227 (1992)381-388. Patent publications describing human antibody phage librariesinchide, for example: U.S. Pat. No. 5,750.373, and US Patent PublicationNos. 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126,2007/0160598, 2007/0237764, 2007/0292936, and 2009/0002360.

Antibodies or antibody fragments isolated from human antibody librariesare considered human antibodies or human antibody fragments herein.

Multispecific Antibodies

In certain embodiments, an antibody provided herein is a multispecificantibody, e.g. a bispecific antibody. Multispecific antibodies aremonoclonal antibodies that have binding specificities for at least twodifferent sites. In certain embodiments, one of the bindingspecificities is for a specific antigen and the other is for any otherantigen. In certain embodiments, bispecific andbodies may bind to twodifferent epitopes of the antigen. Bispecific antibodies may also beused to localize cytotoxic agents to cells which express the antigen towhich the antibody binds. Bispecific antibodies can be prepared as fulllength antibodies or antibody fragments.

Techniques for making multispecific antibodies include, but are notlimited to, recombinant co-expression of two immunoglobulin heavychain-light chain pairs having different specificities (see Milstein,and Cuello, Nature 305 (1983) 537, WO 93/08829, and Traunecker, et al.,EMBO J. 10 (1991) 3655), and “knob-in-hole” engineering (see, e.g., U.S.Pat. No. 5,731,168). Multi-specific antibodies may also be made byengineering electrostatic steering effects for making antibodyFc-heterodimerie molecules (WO 2009/089004 A1); cross-linking two ormore antibodies or fragments (see, e.g ., U.S. Pat. No. 4,676,980, andBrennan, et al., Science, 229 (1985) 81); using leucine zippers toproduce hi-specific antibodies (see, e.g., Kostelny, et al., J.Immunol., 148(5) (1992) 1547-1553); using “diabody” technology formaking bispecific antibody fragments (see, e.g., Hollinger, et al.,Proc. Natl. Acad. Sci. USA, 90 (1993) 6444-6448); and using single-chainFv (sFv) dimers (see, e.g. Gruber, et al., J. Immunol., 152 (1994)5368); and preparing trispecific antibodies as described, e.g., in Tutt,et al., J. Immanol. 147 (1991) 60.

Engineered antibodies with three or more functional antigen bindingsites, including “Octopus antibodies,” are also included herein (see,e.g. US 2006/0025576 A1).

The antibody or fragment herein also includes a “Doal Acting FAb” or“DAR” comprising an antigen binding site that binds to a specificantigen as well as another, different antigen (see, US 2008/0069820, forexample).

Antibody Variants with Altered Binding Affinity to the Antigen

In certain embodiments, it may be desirable to improve the bindingaffinity to the antigen and/or other biological properties of theantibody. Amino acid sequence variants of an antibody may be prepared byintroducing appropriate modifications into the nucleotide sequenceencoding the antibody, or by peptide synthesis. Such modificationsinclude, for example, deletions from, and/or insertions into and/orsubstitutions of residues within the amino acid sequences of theantibody. Any combination of deletion, insertion, and substitution canbe made to arrive at the final construct, provided that the finalconstruct possesses the desired characteristics, e.g., antigen-binding.

Substitution, Insertion, and Deletion Variants

In certain embodiments, polypeptides comprising Fc variants additionallyhave one or more amino acid substitutions at other parts than the Fcpart, are provided. Sites of interest for substitutional mutagenesisinclude the HVRs and FRs. Conservative substitutions are shown in Table1 under the heading of “conservative substitutions.”More substantialchanges are provided in Table 1 under the heading of “exemplarysubstitutions,” and as further described below in reference to aminoacid side chain classes. Amino acid substitutions may be introduced intoan antibody of interest and the products screened for a desiredactivity, e.g., retained/improved antigen binding, or decreasedimmunogenicity.

TABLE 1 Original Conservative Residue Exemplary SubstitutionsSubstitutions Ala (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn(N) Gln; His; Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; AlaSer Gln (Q) Asn; Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H)Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met; Ala; Phe; Norleucine LeuLeu (L) Norleucine; Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; AsnArg Met (M) Leu; Phe; Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr TyrPro (P) Ala Ala Ser (S) Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; PheTyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V) Ile; Leu; Met; Phe; Ala;Norleucine Leugrouped according to common side-chain properties:

-   -   (1) hydrophobic: Ile, Met, Ala, Val, Len, Ile;    -   (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;    -   (3) acidic: Asp, Glu;    -   (4) basic: His, Lys, Arg;    -   (5) residues that influence chain orientation: Gly, Pro;    -   (6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

One type of substitutional variant involves substituting one or morehypervariable region residues of a parent antibody (e.g. a humanized orhuman antibody). Generally, the resulting variant(s) selected forfurther study will have modifications (e.g., improvements) in certainbiological properties (e.g., increased affinity, reduced immunogenicity)relative to the parent antibody and/or will have substantially retainedcertain biological properties of the parent antibody. An exemplarysubstitutional variant is an affinity matured antibody, which may beconveniently generated, e.g., using phage display-based affinitymaturation techniques such as those described herein. Briefly, one ormore HVR residues are mutated and the variant antibodies displayed onphage and screened for a particular biological activity (e.g. bindingaffinity).

Alterations (e.g., substitutions) may be made in HVRs, e.g., to improveantibody affinity. Such alterations may be made in HVR “hotspots,” i.e.,residues encoded by codons that undergo mutation at high frequencyduring the somatic maturation process (see, e.g., Chowdhury, MethodsMol. Biol. 207 (2008) 179-196), and/or SDRs (a-CDRs), with the resultingvariant VH or VL being tested for binding affinity. Affinity maturationby constructing and reselecting from secondary libraries has beendescribed, e.g., in Hoogenboom, et al., in Methods in Molecular Biology178 (2002) 1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)).In. some embodiments of affinity maturation, diversity is introducedinto the variable genes chosen for matoration by any of a variety ofmethods (e.g., error-prone PCR, chain shuffling, oroligonucleotide-directed mutagenesis). A secondary library is thencreated. The library is then screened to identify any antibody variantswith the desired affinity. Another method to introduce diversityinvolves HVR-directed. approaches, in which several HVR residues (e.g.,4-6 residues at a time) are randomized. HVR residues involved in antigenbinding may be specifically identified, e.g., using alanine scanningmutagenesis or modeling. CDR-H3 and CDR-L3 in particular are oftentargeted.

In certain embodiments, substitutions, insertions, or deletions mayoccur within one or more HVRs so long as such alterations do notsubstantially reduce the ability of the antibody to bind antigen. Forexample, conservative alterations (e.g., conservative substitutions asprovided herein) that do not substantially reduce binding affinity maybe made in HVRs. Such alterations may be outside of HVR “hotspots” orSDRs. In certain embodiments of the variant VH and VL sequences providedabove, cach HVR either is unaltered, or contains no more than one, twoor three amino acid substitutions.

A useful method for identification of residues or regions of an antibodythat may be targeted for mutagenesis is called “alanine scanningmutagenesis” as described by Cunningham, and Wells, Science 244 (1989)1081-1085. In this method, a residue or group of target residues (e.g.,charged residues such as arg. asp, his, lys, and glu) are identified andreplaced by a neutral or negatively charged amino acid (e.g., alanine orpolyalanine) to determine whether the interaction of the antibody withantigen is affected. Further substitutions may be introduced at theamino acid locations demonstrating functional sensitivity to the initialsubstitutions. Alternatively, or additionally, a crystal structure of anantigen-antibody complex to identify contact points between the antibodyand antigen. Such contact residues and neighboring residues may betargeted or eliminated as candidates for substitution. Variants may bescreened to determine whether they contain the desired properties.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue. Other insertionalvariants of the antibody molecule inchide the fusion to the N. orC-terminus of the antibody to an enzyme (e.g. for ADEPT) or apolypeptide which increases the serum half-life of the antibody.

Glycosylation Variants

In certain embodiments, an antibody provided herein is altered toincrease or decrease the extent to which the antibody is glycosylated.Addition or deletion of glycosylation sites to an antibody may beconveniently accomplished by altering the amino acid sequence such thatone or more glycosylation sites is created or removed.

Where the antibody comprises an Fc region, the carbohydrate attachedthereto may be altered. Native antibodies produced by mammalian cellstypically comprise a branched, hiantennary oligosaccharide that isgenerally attached by an N-linkage to Asn297 of the CH2 domain of the Fcregion. See, e.g., Wright, et al., TIBTECH 15 (1997) 26-32. Theoligosaccharide may include various carbohydrates, e.g., mannose,N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as afucose attached to a GlcNAc in the “stem” of the biantennaryoligosaccharide structure. In some embodiments, modifications of theoligosaccharide in an antibody of the invention may be made in order tocreate antibody variants with certain improved properties.

Polypeptides comprising Fc variants are further provided with sialylatedoligosaccharides, e.g., in which a differential sialylation of the Fccore oligosaccharide attached to the Fc region of the antibody isprovided. Such polypeptides may have increased sialylation and/ordecreased ADCC function. Examples of such antibody variants aredescribed e.g. by Kaneko, et al., Science 313 (2006) 670-673.

Cysteine Engineered Antibody Variants

In certain embodiments, it may be desirable to create cysteineengineered antibodies, e.g., “thioMAbs,” in which one or more residuesof an antibody are substituted with cysteine residues. In particularembodiments, the substituted residues occur at accessible sites of theantibody. By substituting those residues with cysteine, reactive thiolgroups are thereby positioned at accessible sites of the antibody andmay be used to conjugate the antibody to other moieties, such as drugmoieties or linker-drug moieties, to create an immunoconjugate, asdescribed further herein. In certain embodiments, any one or more of thefollowing residues may be substituted with cysteine: V205 (Kabatnumbering) of the light chain; A118 (EU numbering) of the heavy chain;and S400 (EU numbering) of the heavy chain Fc region. Cysteineengineered antibodies may be generated as described, e.g., in U.S. Pat.No. 7,521,541.

Antibody Derivatives

In certain embodiments, an antibody provided herein may be furthermodified to contain additional nonproteinaceous moieties that are knownin the art and readily available, The moieties suitable forderivatization of the antibody include but are not limited to watersoluble polymers. Non-limiting examples of water soluble polymersinclude, but are not limited to, polyethylene glycol (PEG), copolymersof ethylene glycol/propylene glycol, carboxymethylcellulose, dextran,polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane,poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids(either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol homopolymers,prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylatedpolyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.Polyethylene glycol propionaldehyde may have advantages in manufacturingdue to its stability in water. The polymer may be of any molecularweight, and may be branched or unbranched. The number of polymersattached to the antibody may vary, and if more than one polymer areattached, they can be the same or different molecules. In general, thenumber and/or type of polymers used for derivatization can be determinedbased on considerations including, but not limited to, the particularproperties or functions of the antibody to be improved, whether theantibody derivative will be used in a therapy under defined conditions,etc.

In another embodiment, conjugates of an antibody and nonproteinaceousmoiety that may be selectively heated by exposure to radiation areprovided. In one embodiment, the nonproteinaceous moiety is a carbonnanotube (Kam, et al., Proc. Natl. Acad. Sci. USA 102 (2005)11600-11605). The radiation may be of any wavelength, and includes, butis not limited to, wavelengths that do not harm ordinary cells, butwhich heat the nonproteinaceous moicty to a temperature at which cellsproximal to the antibody-nonproteinaceous moiety are killed.

Recombinant Methods and Compositions

Antibodies may be produced using recombinant methods and compositions,e.g., as described in U.S. Pat. No. 4,816,567. In one embodiment,isolated nucleic acid encoding an antibody variant described herein isprovided. Such nucleic acid may encode an amino acid sequence comprisingthe VL and/or an amino acid sequence comprising the VH of the antibody(e.g., the light and/or heavy chains of the antibody). In a furtherembodiment, one or more vectors (e.g., expression vectors) comprisingsuch nucleic acid are provided. In a further embodiment, a host cellcomprising such nucleic acid is provided. In one such embodiment, a hostcell comprises (e.g., has been transformed with): (1) a vectorcomprising a nucleic acid that encodes an amino acid sequence comprisingthe VL of the antibody and an amino acid sequence comprising the VH ofthe antibody, or (2) a first vector comprising a nucleic acid thatencodes an amino acid sequence comprising the VL. of the antibody and asecond vector comprising a nucleic acid that encodes an amino acidsequence comprising the VH of the antibody. In one embodiment, the hostcell is eukaryotic, e.g. a Chinese Hamster Ovary (CHO) cell or lymphoidcell (e.g., Y0, NS0, Sp20 cell). In one embodiment, a method of makingan antibody variant is provided, wherein the method comprises culturinga host cell comprising a nucleic acid encoding the antibody, as providedabove, under conditions suitable for expression of the antibody, andoptionally recovering the antibody from the host cell (or host cellculture medium).

For recombinant production of an antibody variant, nucleic acid encodingan antibody, e.g., as described above, is isolated and inserted into oneor more vectors for further cloning and/or expression in a host cell.Such nucleic acid may be readily isolated and sequenced usingconventional procedures (e.g., by using oligonucleotide probes that arecapable of binding specifically to genes encoding the heavy and lightchains of the antibody).

Suitable host cells for cloning or expression of antibody-encodingvectors include prokaryotic or eukaryotic cells described herein. Forexample, antibodies may be produced in bacteria, in particular whenglycosylation and Pe effector function are not needed. For expression ofantibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat.Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods inMolecular Biology 248 (2003) 245-254 (B.K.C. Lo, ed., Humana Press,Totowa, NJ), describing expression of antibody fragments in E. coli.)After expression, the antibody may be isolated from the bacterial cellpaste in a soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors, including fungi and yeast strains whoseglycosylation pathways have been “humanized,” resulting in theproduction of an antibody with a partially or fully human glycosylationpattern. See Gerngross, Nat. Biotech. 22 (2004) 1409-1414, and Li, etal., Nat. Biotech. 24 (2006) 210-215.

Suitable host cells for the expression of glycosylated antibody are alsoderived from multicellular organisms (invertebrates and vertebrates).Examples of invertebrate cells include plant and insect cells. Numerousbaculoviral strains have been identified which may be used inconjunction with insect cells, particularly for transfection ofSpodoptera frugiperda cells.

Plant cell cultures can also be utilized as hosts. See, e.g., U.S. Pat.Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429(describing PLANTIBODIES™M technology for producing antibodies intransgenic plants).

Vertebrate cells may also be used as hosts. For example, mammalian celllines that are adapted to grow in suspension may be useful. Otherexamples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7); human embryonic kidney line (293 or kidneycells (BHK); mouse sertoli cells (TM4 cells as described, e.g., inMather, Biol, Reprod. 23 (1980) 243-251); monkey kidney cells (CVI);African green monkey kidney cells (VERO-76); human cervical carcinomacells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL3A); human lung cells (W138); human liver cells (Hep G2); mouse mammarytumor (MMT 060562); TRI cells, as described, e.g., in Mather, et al.,Annals N.Y. Acad. Sci. 383 (1982) 44-68; MRC 5 cells; and FS4 cells.Other useful mammalian host cell lines include Chinese hamster ovary(CHO) cells, including DHFR′ CHO cells (Urlaub, et al., Proc. Natl.Acad. Sci. USA 77 (1980) 4216); and myeloma cell lines such as Y0, NS0and Sp2/0. For a review of certain mammalian host cell lines suitablefor antibody production, see, e.g., Yazaki, and Wu, Methods in MolecularBiology 248 (2003) 255-268 (B.K.C. Lo, ed., Humana Press, Totowa, NJ).

Assays

Antibodies provided herein may be identified, screened for, orcharacterized for their physical/chemical properties and/or biologicalactivities by various assays known in the art.

Binding Assays and Other Assays

In one aspect, an antibody of the invention is tested for its antigenbinding activity. e.g., by known methods such as ELISA, Western blot,etc.

In an exemplary competition assay, immobilized antigen is incubated in asolution comprising a first labeled antibody that binds to the antigen(e.g.,) and a second unlabeled antibody that is being tested for itsability to compete with the first antibody for binding to the antigen.The second antibody may be present in a hybridoma supernatant. As acontrol, immobilized antigen is incubated in a solution comprising thefirst labeled antibody but not the second unlabeled antibody. Afterincubation under conditions permissive for binding of the first antibodyto the antigen, excess onbound antibody is removed, and the amount oflabel associated with immobilized antigen is measured. If the amount oflabel associated with immobilized antigen is substantially reduced inthe test sample relative to the control sample, then that indicates thatthe second antibody is competing with the first antibody for binding tothe antigen (See Harlow, and Lane (1988) Antibodies: A Laboratory Manualch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

Immunoconjugates

The invention also provides immunoconjugates comprising an antibodyherein conjugated to one or more cytotoxic agents, such aschemotherapeutic agents or drugs, growth inhibitory agents, toxins(e.g., protein toxins, enzymatically active toxins of bacterial, fungal,plant, or animal origin, or fragments thereof), or radioactive isotopes.

In one embodiment, an immunoconjugate is an antibody-drug conjugate(ADC) in which an antibody is conjugated to one or more drugs, includingbut not limited to a maytansinoid (see U.S. Pat. Nos. 5,208,020,5,416,064 and European Patent BP 0 425 235 B1); an autistatin such asmonomethylauristatin drug moieties DE and DF (MMAE and MMAF) (see U.S.Pat. Nos. 5,635,483 and 5,780,588, and 7,498,298); a dolastatin; acalicheamicin or derivative thereof (see U.S. Pat. Nos. 5,712,374,5,714,586, 5,739, 116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and5,877,296; Hinman, et al., Cancer Res. 53 (1993) 3336-3342; and Lode, etal., Cancer Res. 58 (1998) 2925-2928); an anthracycline such asdaunomycin or doxorubicin (see Kratz, et al., Current Med. Chem. 13(2006) 477-523; Jeffrey, et al., Bioorganic & Med. Chem. Letters 16(2006) 358-362; Torgov, et al., Bioconj. Chem. 16 (2005) 717-721; Nagy,et al., Proc. Natl. Acad. Sci. USA 97 (2000) 829-834; Dubowchik, et al.,Bioorg. & Med. Chem. Letters 12 (2002) 1529-1532; King, et al., J. Med.Chem. 45 (2002) 4336-4343; and U.S. Pat. No. 6,630,579); methotrexate;vindesine; a taxane such as docetaxel, paclitaxel, larotaxel, tesetaxel,and ortataxel; a trichothecene; and CC1065.

In another embodiment, an immunoconjugate comprises an antibody asdescribed herein conjugated to an enzymatically active toxin or fragmentthereof, including but not limited to diphtheria A chain, nonbindingactive fragments of diphtheria toxin, exotoxin A chain (from Pseudomonasaeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleorites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S). momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.

In another embodiment, an immunoconjugate comprises an antibody asdescribed herein conjugated to a radioactive atom to form aradioconjugate. A variety of radioactive isotopes are available for theproduction of radioconjugates. Examples include At²⁷¹, I¹³¹, I¹²⁵, Y⁹⁰,Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu.When the radioconjugate is used for detection, it may comprise aradioactive atom for scintigraphie studies, for example te99m or 1123,or a spin label for nuclear magnetic resonance (NMR) imaging (also knownas magnetic resonance imaging, mri), such as iodine-123 again,iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17,gadolinium, manganese or iron.

Conjugates of an antibody and cytotoxic agent may be made using avariety of bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-malcimidomethyl) cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azide compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta, et al., Science 238 (1987) 1098.Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO 94/11026. Thelinker may be a “cleavable linker” facilitating release of a cytotoxicdrug in the cell. For example, an acid-labile linker,peptidase-sensitive linker, photolabile linker, dimethyl linker ordisulfide-containing linker (Chari, et al, Cancer Res. 52 (1992)127-131; U.S. Pat. No. 5,208,020) may be used.

The immunnoconjugates or ADCs herein expressly contemplate, but are notlimited to such conjugates prepared with cross-linker reagentsincluding, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS,MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS,sulfo-KMUS, sulfo-MBS. suffo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB(succinimidyl-(4-vinylsulfone)benzoate) which are commercially available(e.g., from Pierce Biotechnology, Inc., Rockford, IL., U.S.A).

Methods and Compositions for Diagnostics and Detection

In certain embodiments, any of the antibody variants provided herein isuseful for detecting the presence of the antigen binding to thatantibody in a biological sample. The term “detecting” as used hereinencompasses quantitative or qualitative detection. In certainembodiments, a biological sample comprises a cell or tissuc.

In one embodiment, an antibody variant for use in a method of diagnosisor detection is provided. In a further aspect, a method of detecting thepresence of the antigen to which said antibody variant binds in abiological sample is provided. In certain embodiments, the methodcomprises contacting the biological sample with an antibody as describedherein under conditions permissive for binding of the antibody to theantigen, and detecting whether a complex is formed between the antibodyand the antigen. Such method may be an in vitro or in vivo method. Inone embodiment, an antibody variant is used to select subjects eligiblefor therapy with an antibody, e.g. where the antigen to which saidantibody binds is a biomarker for selection of patients.

Exemplary disorders that may be diagnosed using an antibody of theinvention include cancer, cardiovascular diseases, neuronal disordersand diabetes.

In certain embodiments, labeled antibody variants are provided. Labelsinclude, but are not limited to, labels or moieties that are detecteddirectly (such as fluorescent, chromophoric, electron-dense,chemiluminescent, and radioactive labels), as well as moieties, such asenzymes or ligands, that are detected indirectly, e.g., through anenzymatic reaction or molecular interaction. Exemplary labels include,but are not limited to, the radioisotopes ³²P, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I,fluorophores such as rare earth chelates or fluorescein and itsderivatives, rbodamine and its derivatives. dansyl, umbelliferone,luceriferases, e.g., firefly luciferase and bacterial luciferase (U.S.Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones,borseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase,glucoamylase, lysozyme, saccharide oxidases, e.g., glucose oxidase,galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclicoxidases such as uricase and xanthine oxidase, coupled with an enzymethat employs hydrogen peroxide to oxidize a dye precursor such as HRP,lactoperoxidase, or microperoxidase, biotin/avidin, spin labels,bacteriophage labels, stable free radicals, and the like.

Pharmaceutical Formulations

Pharmaceutical formulations of an antibody variant as described hereinare prepared by mixing such antibody having the desired degree of puritywith one or more optional pharmaceutically acceptable carriers(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)),in the form of lyophilized formulations or aqueous solutions.Pharmaceutically acceptable carriers are generally nontoxic torecipients at the dosages and concentrations employed, and include, butare not limited to: buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride; benzethoniom chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-eresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immanoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trebalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Za-proteincomplexes); and/or non-ionic surfactants such as polyethylene glycol(PEG). Exemplary pharmaceutically acceptable carriers herein furtherinclude interstitial drug dispersion agents such as solubleneutral-active hyaluronidase glycoproteins (sHASEGP), for example, humansoluble PH-20 hyaluronidase glycoproteins, such as fHuPH20 (HYLENEX®,Baxter International. Inc.). Certain exemplary sHASEGPs and methods ofuse, including rHaPH20, are described in US Patent Publication Nos.2005/0260186 and 2006/0104968. In one aspect. a SHASEGP is combined withor one more additional glycosaminoglycanases such as chondroitinases.

Exemplary lyophilized antibody formulations are described in U.S. Pat.No. 6,267,958. Aqueous antibody formulations include those described inU.S. Pat. No. 6,171,586 and WO 2006/044908, the latter formulationsincluding a histidine-acetate buffer.

The formulation herein may also contain more than one active ingredientsas necessary for the particular indication being treated, preferablythose with complementary activities that do not adversely affect eachother. Such active ingredients are suitably present in combination inamounts that are effective for the purpose intended.

Active ingredients may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules.

The formulations to be used for in vivo administration are generallysterilo. Sterility may be readily accomplished, e.g., by filtrationthrough sterile filtration membranes.

Therapeutic Methods and Compositions

Any of the polypeptides provided herein may be used in therapeuticmethods.

In a specific aspect of the invention the polypeptide according to theinvention are used for treating a disease. In a more specific aspect,the disease is such, that it is favorable that the effector function ofthe variant is strongly, at least by 50%. reduced compared to thepolypeptide comprising the wildtype Fc polypeptide.

In a specific aspect the polypeptide according to the invention is usedin the manufacture of a medicament for the treatment of a disease,wherein it is favorable that the effector function of the polypeptide isstrongly reduced compared to a wildtype Fc polypeptide. In a furtherspecific aspect the polypeptide according to the invention is used inthe manufacture of a medicament for the treatment of a disease, whereinit is favorable that the effector function of the polypeptide is reducedcompared to a wildtype Fc polypeptide, by at least 20%.

A further aspect is a method of treating an individual having a disease,wherein it is favorable that the effector function of the variant isstrongly reduced compared to a wildtype Fc polypeptide, comprisingadministering to the individual an effective amount of the polypeptideaccording to the invention.

A strong reduction of effector fonction is a reduction of effectorfunction by at least 50% of the effector function induced by thewildtype polypeptide.

Such diseases are for example all diseases where the targeted cellshould not be destroyed by for example ADCC, ADCP or CDC. Moreover, thisis true for those antibodies that are designed to deliver a drug (c.g.,toxins and isotopes) to the target cell where the Fc/FcγR mediatedeffector functions bring healthy immune cells into the proximity of thedeadly payload, resulting in depletion of normal lymphoid tissue alongwith the target cells (Hutchins, et al, PNAS USA 92 (1995) 11980-11984;White, et al, Annu Rev Med 52 (2001) 125-145). In these cases the use ofantibodies that poorly recruit complement or effector cells would be oftremendous benefit (scc for example, Wa, et al., Cell Immunol 200 (2000)16-26; Shields, et al., J. Biol Chen 276(9) (2001) 6591-6604; U.S. Pat.Nos. 6,194,551; 5,885,573 and PCT publication WO 04/029207).

In other instances, for example, where blocking the interaction of awidely expressed receptor with its cognate ligand is the objective, itwould be advantageous to decrease or eliminate all antibody effectorfunction to reduce unwanted toxicity. Also, in the instance where atherapeutic antibody exhibited promiscuous binding across a number ofhuman tissues it would be prudent to limit the targeting of effectorfunction to a diverse set of tissues to limit toxicity.

Also for agonist antibodies it would be very helpful if these antibodiesexhibit reduced effector function.

The conditions which can be treated with the polypeptide variant aremany and include cancer (e.g. where the antibody variant binds the HER2receptor, angiopoietin receptor or vascular endothelial growth factor(VEGF)); allergic conditions such as asthma (with an and-IgE antibody);and LFA-1-mediated disorders (e.g. where the polypeptide variant is ananti-LFA-1 or anti-ICAM-1 antibody), neurological and metabolicdisorders.

Where the antibody binds the HER2 receptor, the disorder preferably isHER2-expressing cancer, e.g. a benign or malignant tumor characterizedby overexpression of the HER2 receptor. Such cancers include, but arenot limited to, breast cancer, squamous cell cancer, small-cell lungcancer, non-small cell lung cancer, gastrointestinal cancer, pancreaticcancer, glioblastoma, cervical cancer, ovarian cancer, bladder cancer,hepatoma, colon cancer, colorectal cancer, endometrial carcinoma,salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer,vulval cancer, thyroid cancer, hepatic carcinoma and various types ofhead and neck cancer.

The polypeptide or antibody variant is administered by any suitablemeans, including parenteral, subcutaneous, intraperitoneal,intrapulmonary, and intranasal, and, if desired for localimmunosuppressive treatment, intralesional administration. Parenteralinfusions include intramuscular, intravenous, intraarterial.intraperitoneal, or subcutaneous administration. In addition, theantibody variant is suitably administered by pulse infusion,particularly with declining doses of the polypeptide variant. Preferablythe dosing is given by injections, most preferably intravenous orsubcutaneous injections, depending in part on whether the administrationis brief or chronic.

For the prevention or treatment of disease, the appropriate dosage ofpolypeptide or antibody variant will depend on the type of disease to betreated, the severity and course of the disease, whether the polypeptidevariant is administered for preventive or therapeutic purposes, previoustherapy, the patient's clinical history and response to the polypeptidevariant, and the discretion of the attending physician, The polypeptidevariant is suitably administered to the patient at one time or over aseries of treatments.

Depending on the type and severity of the disease, about 1 μg/kg to 15mg/kg (e.g., 0.1-20 mg/kg) of polypeptide or antibody variant is aninitial candidate dosage for administration to the patient, whether, forexample, by one or more separate administrations, or by continuousinfusion. A typical daily dosage might range from about 1 μg/kg to 100mg/kg or more, depending on the factors mentioned above. For repeatedadministrations over several days or longer, depending on the condition,the treatment is sustained until a desired suppression of diseasesymptoms occurs. However, other dosage regimens may be useful. Theprogress of this therapy is easily monitored by conventional techniquesand assays.

In certain embodiments, the invention provides an antibody variant orpolypeptide for use in a method of treating an individual having cancercomprising administering to the individual an effective amount of theantibody variant. In one such embodiment, the method farther comprisesadministering to the individual an effective amount of at least oneadditional therapeutic agent, e.g., as described below. In furtherembodiments, the invention provides an antibody variant for use ininhibiting angiogenesis, inhibiting cell proliferation or depletingB-cells in an individual comprising administering to the individual aneffective of the antibody variant to inhibit angiogenesis, inhibit cellproliferation or deplete B-cells in an “individual” according to any ofthe above embodiments is preferably a human.

In a further aspect, the invention provides for the use of an antibodyvariant or polypeptide in the manufacture or preparation of amedicament. In one embodiment, the medicament is for treatment of canceror inflammatory diseases. In a further embodiment, the medicament is foruse in a method of treating cancer, diabetes, neuronal disorders orinflammatory comprising administering to an individual having cancer,diabetes, neuronal disorders or inflammatory an effective amount of themedicament. In one such embodiment, the method further comprisesadministering to the individual an effective amount of at least oneadditional therapeutic agent, e.g., as described below. In a furtherembodiment, the medicament is for inhibiting angiogenesis, inhibitingcell proliferation or depleting B-cells.

In a further embodiment, the medicament is for use in a method ofinhibiting angiogenesis, inhibiting cell proliferation or depletingB-cells in an individual comprising administering to the individual anamount effective of the medicament to inhibit angiogenesis, inhibit cellproliferation or deplete B-cells. An “individual” according to any ofthe above embodiments may be a human.

In a further aspect, the invention provides pharmaceutical formulationscomprising any of the antibody variants provided herein, e.g., for usein any of the above therapeutic methods. In one embodiment, apharmaceutical formulation comprises any of the antibody variantsprovided herein and a pharmaceutically acceptable carrier. In anotherembodiment, a pharmaceutical formulation comprises any of the antibodyvariants provided herein and at least one additional therapeutic agent,e.g., as described below.

Antibodies of the invention con be used either alone or in combinationwith other agents in a therapy. For instance, an antibody of theinvention may be co-administered with at least one additionaltherapeutic agent.

Such combination therapies noted above encompass combined administration(where two or more therapeutic agents are included in the same orseparate formulations), and separate administration, in which case,administration of the antibody of the invention can occur prior to,simultaneously, and/or following, administration of the additionaltherapeutic agent and/or adjuvant. Antibodies of the invention can alsobe used in combination with radiation therapy.

An antibody of the invention (and any additional therapeutic agent) canbe administered by any suitable means, including parenteral,intrapulmonary, and intranasal, and, if desired for local treatment,intralesional administration, Parenteral infusions includeintramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. Dosing can be by any suitable route, e.g.by injections, such as intravenous or subcutaneous injections, dependingin part on whether the administration is brief or chronic. Variousdosing schedules including but not limited to single or multipleadministrations over various time-points, bolus administration, andpulse infusion are contemplated herein.

Antibodies of the invention would be formulated, dosed, and administeredin a fashion consistent with good medical practice. Factors forconsideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. Theantibody need not be, but is optionally formulated with one or moreagents currently used to prevent or treat the disorder in question. Theeffective amount of such other agents depends on the amount of antibodypresent in the formulation, the type of disorder or treatment, and otherfactors discussed above. These are generally used in the same dosagesand with administration routes as described herein, or about from 1 to99% of the dosages described herein, or in any dosage and by any routethat is empirically/clinically determined to be appropriate.

For the prevention or treatment of disease, the appropriate dosage of anantibody of the invention (when used alone or in combination with one ormore other additional therapeutic agents) will depend on the type ofdisease to be treated, the type of antibody, the severity and course ofthe disease, whether the antibody is administered for preventive ortherapeutic purposes, previous therapy, the patient's clinical historyand response to the antibody, and the discretion of the attendingphysician. The antibody is suitably administered to the patient at onetime or over a series of treatments. Depending on the type and severityof the disease, about 1 μg/kg to 15 mg/kg (e.g. 0.1 mg/kg-10mg/kg) ofantibody can be an initial candidate dosage for administration to thepatient, whether, for example, by one or more separate administrations,or by continuous infusion. One typical daily dosage might range fromabout 1 μg/kg to 100 mg/kg or more, depending on the factors mentionedabove. For repeated administrations over several days or longer,depending on the condition, the treatment would generally be sustainedontil a desired suppression of disease symptoms occurs. One exemplarydosage of the antibody would be in the range from about 0.05 mg/kg toabout 10 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg,4.0 mg/kg or 10 mg/kg (or any combination thereof) may be administeredto the patient. Such doses may be administered intermittently, e.g.every week or every three weeks (e.g. such that the patient receivesfrom about two to about twenty, or e.g. about six doses of theantibody). An initial higher loading dose, followed by one or more lowerdoses may be administered. However, other dosage regimens may be useful.The progress of this therapy is easily monitored by conventionaltechniques and assays,

It is understood that any of the above formulations or therapeuticmethods may be carried out using an immunoconjugate of the invention inplace of or in addition to an antibody according to the invention.

Articles of Manufacture

In another aspect of the invention, an article of manufacture containingmaterials useful for the treatment, prevention and/or diagnosis of thedisorders described above is provided. The article of manufacturecomprises a container and a label or package insert on or associatedwith the container. Suitable containers include, for example, bottles,vials, syringes, IV solution bags, etc. The containers may be formedfrom a variety of materials such as glass or plastic. The containerholds a composition which is by itself or combined with anothercomposition effective for treating, preventing and/or diagnosing thecondition and may have a sterile access port (for example the containermay be an intravenous solution bag or a vial baving a stopper pierceableby a hypodermic injection needle). At least one active agent in thecomposition is an antibody of the invention, The label or package insertindicates that the composition is used for treating the condition ofchoice. Moreover, the article of manufacture may comprise (a) a firstcontainer with a composition contained therein, wherein the compositioncomprises an antibody of the invention; and (b) a second container witha composition contained therein, wherein the composition comprises afurther cytotoxic or otherwise therapeutic agent. The article ofmanufacture in this embodiment of the invention may further comprise apackage insert indicating that the compositions can be used to treat aparticular condition. Alternatively, or additionally, the article ofmanufacture may further comprise a second (or third) containercomprising a pharmaceutically-acceptable buffer, such as bacteriostaticwater for injection (BWFI), phosphate-buffered saline, Ringer's solutionand dextrose solution. It may further include other materials desirablefrom a commercial and user standpoint, including other buffers,diluents, filters, needles, and syringes.

It is understood that any of the above articles of manufacture mayinclude an immunoconjugate of the invention in place of or in additionto an antibody variant.

Non-Therapeutic Uses for the Polypeptide

The antibody variant of the invention may be used as an affinitypurification agent. In this process, the antibody variant is immobilizedon a solid phase such a Sephadex resin or filter paper, using methodswell known in the art. The immobilized polypeptide variant is contactedwith a sample containing the antigen to be purified, and thereafter thesupport is washed with a suitable solvent that will remove substantiallyall the material in the sample except the antigen to be purified, whichis bound to the immobilized antibody variant. Finally, the support iswashed with another suitable solvent, such as glycine buffer, pH 5.0,that will release the antigen from the polypeptide variant.

The antibody variant may also be useful in diagnostic assays, e.g., fordetecting expression of an antigen of interest in specific cells,tissues, or serom.

For diagnostic applications, the antibody variant typically will belabeled with a detectable moiety. Numerous labels are available whichcan be generally grouped into the following categories:

-   -   (a) Radioisotopes, such as ³⁵s, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I. The        polypeptide variant can be labeled with the radioisotope using        the techniques described in Coligen, et al., Current Protocols        in Immunology, Volumes 1 and 2, Ed. Wiley-Interscience, New        York, N.Y., Pubs. (1991) for example and radioactivity can be        measured using scintillation counting.    -   (b) Fluorescent labels such as rare earth chelates (europium        chelates) or fluorescein and its derivatives, rhodamine and its        derivatives, dansyl, Lissamine, phycoerythrin and Texas Red are        available. The fluorescent labels can be conjugated to the        polypeptide variant using the techniques disclosed in Current        Protocols in Immunology, supra, for example. Fluorescence can be        quantified using a fluorimeter.    -   (c) Various enzyme-substrate labels are available and U.S. Pat.        No. 4,275,149 provides a review of some of these. The enzyme        generally catalyzes a chemical alteration of the chromogenic        substrate that can be measured asing various techniques. For        example, the enzyme may catalyze a color change in a substrate,        which can be measured spectrophotometrically. Alternatively, the        enzyme may alter the fluorescence or chemiluminescence of the        substrate, Techniques for quantifying a change in fluorescence        are described above. The chemiluminescent substrate becomes        electronically excited by a chemical reaction and may then emit        light which can be measured (using a chemiluminometer, for        example) or donates energy to a fluorescent acceptor. Examples        of enzymatic labels include luciferases (c.g., firefly        luciferase and bacterial luciferase; U.S. Pat. No. 4,737,456),        luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase,        urease, peroxidase such as horseradish peroxidase (HRPO),        alkaline phosphatase, @-galactosidase, glucoamylase, lysozyme,        saccharide oxidases (e.g., glucose oxidase, galactose oxidase,        and glucose-6-phosphate dehydrogenase), heterocyclic oxidases        (such as uricase and xanthine oxidase), lactoperoxidase,        microperoxidase, and the like. Techniques for conjugating        enzymes to antibodies are described in O'Sullivan, et al.,        Methods for the Preparation of Enzyme-Antibody Conjugates for        use in Enzyme Immunoassay, in Methods in Enzym. (ed J. Langone        & H. Van Vunakis), Academic press, New York, 73:147-166 (1981).

Examples of enzyme-substrate combinations include, for example:

-   -   (i) Horseradish peroxidase (HRPO) with hydrogen peroxidase as a        substrate, wherein the hydrogen peroxidase oxidizes a dye        precursor (e.g., orthophenylene diamine (OPD) or        3,3′,5,5′-tetramethyl benzidine hydrochloride (TMB));    -   (ii) alkaline phosphatase (AP) with para-Nitrophenyl phosphate        as chromogenic substrate; and    -   (iii) @-D-galactosidase (B-D-Gal) with a chromogenic substrate        (e.g., p-nitrophenyl-β-D-galactosidase) or fluorogenic substrate        4-methylumbelliferyl-β-D. galactosidase.

Numerous other enzyme-substrate combinations are available to thoseskilled in the art. For a general review of these, see U.S. Pat. Nos.4,275,149 and 4,318,980.

Sometimes, the label is indirectly conjugated with the polypeptidevariant. The skilled artisan will be aware of various techniques forachieving this. For example, the polypeptide variant can be conjugatedwith biotin and any of the three broad categories of labels mentionedabove can be conjugated with avidin, or vice versa.

Biotin binds selectively to avidin and thus, the label can be conjugatedwith the polypeptide variant in this indirect manner. Alternatively, toachieve indirect conjugation of the label with the polypeptide variant.the polypeptide variant is conjugated with a small hapten (e.g.,digoxin) and one of the different types of labels mentioned above isconjugated with an anti-hapten polypeptide variant (e.g., anti-digoxinantibody). Thus, indirect conjugation of the label with the polypeptidevariant can be achieved.

In another embodiment of the invention, the antibody variant need not belabeled, and the presence thereof can be detected using a labeledantibody which binds to the polypeptide variant.

The antibody variant of the present invention may be employed in anyknown assay method, such as competitive binding assays, direct andindirect sandwich assays, and immunoprecipitation assays. Zola,Monoclonal Antibodies: A Manual of Techniques, (1987) pp. 147-158, CRCPress, Inc.

The antibody variant may also be used for in vivo diagnostic assays.Generally, the polypeptide variant is labeled with a radionuclide (suchas ¹¹¹In, ⁹⁹Tc, ¹⁴C, ¹³¹I, ¹²⁵I, ³H, ³²P or ³⁵S) so that the antigen orcells expressing it can be localized using immunoscintiography. Althoughthe foregoing invention has been described in some detail by way ofillustration and example for purposes of clarity of understanding, thedescriptions and examples should not be construed as limiting the scopeof the invention.

Description of the sequence listing: SEQ ID NO: 1 Human kappa lightchain SEQ ID NO: 2 Human lambda light chain SEQ ID NO: 3 Human IgG1(Caucasian Allotype) SEQ ID NO: 4 Human IgG1 (Afroamerican Allotype SEQID NO: 5 Human IgG1 LALA-Mutant (Caucasian Allotype) SEQ ID NO: 6 HumanIgG4 SEQ ID NO: 7 Human IgG4 SPLE-Mutant which represent exemplary humansequences for the kappa light chain, lambda light chain, IgG1 and IgG4which could serve as basis for generating the variants according to theinvention. In sequence Id Nos 3-5, the sequence of human IgG1 allotypes,the P329 region according to Kabat EU index is located at position 212,whereas said P329 region in sequence Id Nos 6 and 7 can be find atposition 209. SEQ ID NO: 8 Kappa light chain of mAb 40A746.2.3 SEQ IDNO: 9 Heavy chain of wildtype IgG1 of mAb 40A746.2.3 SEQ ID NO: 10 Heavychain of IgG1 P329G of mAb 40A746.2.3 SEQ ID NO: 11 Heavy chain of IgG1LALA/P329G of mAb 40A746.2.3 SEQ ID NO: 12 Heavy chain of IgG4 SPLE ofmAb 40A746.2.3 SEQ ID NO: 13 Heavy chain of IgG4 SPLE/P329G of mAb40A746.2.3 SEQ ID NO: 14 Heavy chain of IgG1 LALA of mAb 40A746.2.3

EXAMPLES

The following seven examples are examples of methods and compositions ofthe invention, It is understood that various other embodiments may bepracticed, given the general description provided above,

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. The disclosures of all patent andscientific literature cited herein are expressly incorporated in theirentirety by reference.

Example 1

Antibodies

For the experiments described below antibodies against CD9 (see SEQ IDs8-14), P-selectin (sequences described in WO 2005/100402) and CD20(synonym: GA101, sequences described in EP 1 692 182) were used.

All variants described herein, e.g. P329G, P329A, P329R SPLE, LALA,P329G/LALA, P329G/SPLE variants of the selectin, CD9, CD20 (GA101) andCD20 (GA101)-glycoengineered binding antibody (numbering according to EUnomenclature) were generated using PCR based mutagenesis. IgG moleculeswere expressed in the HEK-EBNA or HEK293 (CD9 Fc variants) system, andpurified using protein A and size exclusion chromatography.

Example 2

Determination of the Binding Affinities of Different FcγReceptors toImmunoglobulins

Binding affinities of different FcγRs towards immunoglobulins weremeasured by Surface Plasmon Resonance (SPR) using a Biacore T100instrument (GE Healthcare) at 25° C.

The BIAcore® system is well established for the study of moleculeinteractions. It allows a continuous real-time monitoring ofligand/analyte bindings and thus the determination of association rateconstants (k_(a)), dissociation rate constants (k_(d)), and equilibriumconstants (K_(D)). Changes in the refractive index indicate mass changeson the surface caused by the interaction of immobilized ligand withanalyte injected in solution. If molecules bind immobilized ligands onthe surface the mass increases, in case of dissociation the massdecreases.

For a 1:1 interaction no difference in the results should be seen if abinding molecule is either injected over the surface or immobilized ontoa surface. Therefore different settings were used (with Fcγ receptor asligand or analyte respectively), depending on solubility andavailability of ligand or corresponding analyte.

For FcγRI 10000 resonance units (RU) of a capturing system recognizing apolyhistidine sequence (pentaHis monoclonal antibody, Qiagen Hilden,cat. no. 34660) was immobilized by the use of an amine coupling kitsupplied by the GB Healthcare and a CM5 chip at pH 4.5. FcγRI wascaptured at a concentration of 5 μg/ml by with a pulse of 60 sec at aflow of 5 μl/min. Different concentrations of antibodies ranging from 0to 100 nM were passed with a flow rate of 30 μl/min through the flowcells at 298 K for 120 sec to record the association phase. Thedissociation phase was monitored for up to 240 sec and triggered byswitching from the sample solution to running buffer. The surface wasregenerated by 2 min washing with a glycine pH 2 solution at a flow rateof 30 ml/min. For all experiments HBS-P+ buffer supplied by GEHealthcare was chosen (10 mM HEPES, pH 7.4, 150 mM NaCl, 0.05% (v/v)Surfactant P20). Bulk refractive index differences were corrected for bysubtracting the response obtained from a surface without captured FcγRI.Blank injections are also subtracted (=double referencing).

The equilibrium dissociation constant (K_(D)), defined as k_(a)/k_(D),was determined by analyzing the sensogram curves obtained with severaldifferent concentrations, using BIAevaluation software package. Thefitting of the data followed a suitable binding model.

For FcγRIIA and PcyRIIIAV158 10000 resonance units (RU) of a monoclonalantibody to be tested was immobilized onto a CMS chip by the use of anamine coupling kit supplied by the GE (pH 4.5 at a concentration of 10μg/ml).

Different concentrations of FcγRIIA and IIIA ranging from 0 to 12800 nMwere passed with a flow rate of 5 μl/min through the flow cells at 298 Kfor 120 see to record the association phase. The dissociation phase wasmonitored for up to 240 sec and triggered by switching from the samplesolution to running buffer. The surface was regenerated by 0.5 minwashing with a 3 mM NaOH/IM NaCl solution at a flow rate of 30 ml/min.For all experiments HBS-P+ buffer supplied by GE Healthcare was chosen(10 mM HEPES, pH 7.4, 150 mM NaCl, 0.05% (v/v) Surfactant P20).

Bulk refractive index differences were corrected for by subtracting theresponse obtained from a surface without captured antibody. Blankinjections are also subtracted (=double referencing).

The equilibrium dissociation constant (K_(D)), was determined byanalyzing the sensogram curves obtained with several differentconcentrations, using BIA evaluation software package. The fitting ofthe data followed a suitable binding model using steady state fitting

For FcγRIIB 10000 resonance units (RU) of a capturing system recognizinga polyhistidine sequence (pentaHis monoclonal antibody, Qiagen Hilden,cat. no. 34660; was immobilized by the use of an amine coupling kitsupplied by the GE Healthcare and a CMS chip at pH 4.5. FcγRIIB wascaptured at a concentration of 5 μg/ml by with a pulse of 120 sec at aflow of 5 μl/min. Different antibodies were passed at a concentration of1340 nM with a flow rate of 5 μl/min through the flow cells at 298 K for60 sec to record the association phase. The dissociation phase

was monitored for up to 120 sec and triggered by switching from thesample solution to running buffer. The surface was regenerated by 0.5min washing with a glycine pH2.5 solution at a flow rate of 30 ml/min.For all experiments HBS-P+ buffer supplied by GE Healthcare was chosen(10 mM HEPES, pH 7.4, 150 mM NaCl, 0.05% (v/v) Surfactant P20).

Bulk refractive index differences were corrected for by subtracting theresponse obtained from a surface without captured FcγRIIB. Blankinjections are also subtracted (=double referencing).

Due to the very low intrinsic affinity of FcγRIIB to wildtype IgG1 noaffinity was calculated rather a qualitative binding was assessed.

The following tables summarize the effects of introducing a mutationinto the Fc part on binding to FcγRI, FcγRIIA, FcγRIIB, and FcγRIIIAV1-58 (A) as well as the effect on ADCC (measured without (BLT) and withtarget cells (ADCC)) and on C1q binding (B)

TABLE 2A FcγRI FcγRIIaR131 FcγRIIIAV158 FcγRIIB WT IgG1 ++ (5 nM) ++ (2μM) + (0.7 μM) ++ IgG4 SPLE − +/− (10 μM) − (>20 μM) + IgG1 P329G ++ (6nM) − (>20 μM) − (>20 μM) − IgGI P329A ++ (8 nM) + (4.4 μM) + (1.8 μM) +ge IgG1 P329G − − (>20 μM) − (>20 μM) − LALA IgGI P329G ++ (10 nM) −(>20 μM) − (>10 μM) − ge *++ for ge IgG1 30 nM

TABLE 2B ADCC ADCC without with target target Mutant FcγRI FcγRIIFcγRIII C1q cells cells Assay Biacore Biacore Biacore CDC C1q BLT ADCCP329G + — — — — — — P329R n.d. n.d. n.d. n.d. n.d. — — LALA − n.d. − −n.d. n.d. — IgG1_P329G/ — — — n.d. n.d. n.d. n.d. LALA IgG4_SPLE — − — —— n.d. n.d. — strongly reduced/inactive in contrast to wt, − reduced incontrast to wt, + comparable to wt interaction, n.d. not determined, /no result

In more detail the following results have been obtained:

Affinity to the FcγRI Receptor

P329G, P329A, SPLE and LALA mutations have been introduced into the Fopolypeptide of a P-selectin, CD20 and CD9 antibody, and the bindingaffinity to FcγRI was measured with the Biacore system. Whereas theantibody with the P329G mutation still binds to FcγR1 (FIG. 1 a and 1b),introduction of triple mutations P329G/LALA and P329G/SPLE,respectively, resulted in antibodies for which nearly no binding couldbe detected (FIG. 1 b ). The LALA or SPLE mutations decreased binding tothe receptor more than P329G alone but less than in combination withP329G (FIG. 1 a and 1 b ). Thus, the combination of P329G with eitherLALA or SPLE mutations is much more effective than the P329G mutation orthe double mutations LALA or SPLE alone. The kd value for the CD20 IgG1wildtype antibody was 4.6 nM and for the P329G mutant of the sameantibody 5.7 nM, but for the triple mutant P329G/LALA no kd value couldbe determined due to the nearly undetectable binding of the antibody tothe FcγRI receptor. The antibody itself, i.e. whether a CD9 or CD20 orP-selectin was tested, has a minor effect on the binding affinities.

Affinity to the FcγRIIA Receptor

P329G, SPLE and LALA mutations, respectively, have been introduced intothe Fe polypeptide of the CD9 antibody and the binding affinity to theFcγRIIA-R131 receptor was measured with the Biacore system. Bindinglevel is normalized such as captured mAb represents 100 RU. So not morethan approximately 20 RU is expected for a 1:1 stoichiometry. FIG. 1 eshows that the binding to the FcγRIIA receptor is strongly reduced byintroducing the LALA, SPLE/P329G, P329G and LALA/329G mutation into theFc variant. In contrast to binding to the FcγR1 receptor, theintroduction of the P329G mutation alone is able to very strongly blockthe binding to said receptor, more or less to a similar extent as thetriple mutation P329G/LALA (FIG. 1 c ).

Affinity to the FcγRIIB Receptor

SPLE, LALA, SPLE/P329G and LALA/P329G mutations, respectively, bave beenintroduced into the Fc polypeptide of the CD9 and P-selectin antibodyand the binding affinity to FcγRIIB receptor was measured with theBiacore system. FIG. 1 d shows that the binding to the FcγRIIB receptoris strongly reduced in the LALA and triple mutants P329G/LALA,P329G/SPLB

Affinity to the FcγRIIIA Receptor

P329G,LALA, SPLE, P329G/LALA, and SPLE/P3290 mutations have been.introduced into the Fc polypeptide of the CD9 and the binding affinityto FcγRIIIA-V158 receptor was measured with the Biacore system. TheP329G mutation and the triple mutation P329G/LALA reduced binding to theFcγRIIIA receptor most strongly, to nearly undetectable levels. TheP329G/SPLE also lead to a strongly reduced binding affinity, themutations SPLE and LALA, respectively, only slightly decreased thebinding affinity to the FcγRIIIA receptor (FIG. 1 e ).

Example 3

C1Q ELISA

The binding properties of the different polypeptides comprising Fcvariants to C1q were analyzed by an ELISA sandwich type immunoassay.Each variant is coupled to a hydrophobic Maxisorp 96 well plate at 8concentrations between 10 μg/ml and 0 μg/ml. This coupling simulatescomplexes of antibodies, which is a prerequisite for high affinitybinding of the C1q molecule. After washing, the samples are incubated toallow C1q binding. After further washing the bound C1q molecule isdetected by a polyclonal rabbit anti-hC1q antibody. Following the nextwashing step, an enzyme labelled anti-rabbit-Fcγspecific antibody isadded, Immunological reaction is made visible by addition of a substratethat is converted to a coloured product by the enzyme. The resultingabsorbance, measured photometrically, is proportional to the amount ofC1g bound to the antibody to be investigated. EC₅₀ values of thevariant- C1q interaction were calculated. The absorption units resultingfrom the coloring reaction are plotted against the concentration of theantibody. The antibody concentration at the half maximum responsedetermines the ECso value. This read-out is reported as relativedifference to the reference standard measured on the same plate togetherwith the coefficient of variation of sample and reference.

The P329G mutation introduced into the P-selectin or CD20 antibodystrongly reduced binding to C1q, similar to the SPIB mutation (FIG. 2 ).Table 3 summarizes the calculated EC 50 values for binding of thevariants to the C1q receptor. C1q belongs to the complement activationproteins and plays a major role in the activation of the classicalpathway of the complement, which leads to the formation of the membraneattack complex. C1q is also involved in other immunological processessuch as enhancement of phagocytosis, clearance of apoptotic cells orneutralization of virus. Thus, it can be expected that the mutants shownhere to reduce binding to C1q, e.g. P329G and SPLE, as well as verylikely also the triple mutations comprising the aforementioned singlemutations, strongly reduces the above mentioned functions of C1q.

TABLE 3 Antibody EC50 value P-Selectin IgG1 wt 1.8 GA101 IgG1 wt 2.4P-Selectin IgG1_P329G 2.7 P-Selectin IgG4 SPLE 3.0 GA101 IgG1 P329G 5.5GA101 IgG4 SPLE >10

Example 4

ADCC without Target Cells, BLT Assay

The antibodies to be tested (CD20 (GA101) and CD9) were coated in PBSover night at 4° C. in suitable 96-flat bottom well plates. Afterwashing the plate with PBS, the remaining binding sites were blockedwith PBS/1% BSA solution for 1 h at RT. In the meantime, the effectorcells (NK-92 cell line transfected to express low or high affine humanFcγRIII) were harvested and 200 000 living cells/well were seeded in 100μl/well AIM V medium into the wells after discarding the blockingbuffer. 100 μl/well saponin buffer (0.5% saponin+1% BSA in PBS) was usedto determine the maximal esterase release by the effector cells. Thecells were incubated for 3 h at 37° ° C., 5% CO₂ in a incubator. After 3h, 20 pl/well of the supernatants were mixed with 180 μl/well BLTsubstrate (0.2 mM BLT+0.11 mM DTNB in 0.1 M Tris-HCL, pH 8.0) andincubated for 30 min at 37° C. before reading the plate at 405 nm in amicroplate reader. The percentage of esterase release was determinedsetting the maximal release (saponin treated cells) to 100% and theunstimulated cells (no ab coated) to 0% release.

The wildtype CD20 antibody (GA101 wt (1)) shows strong induction ofcytolytic activity. The LALA variant shows a marked reduction inesterase release, whereas the P3290 and the P329G/LALA variant do notshow any ADCC activity (FIG. 3 a ). FIG. 3 b shows that not only anexchange of G at position P329 leads to markedly reduced cytosolicactivity but also an exchange of P329 to R329 (CD20 antibody). Thusarginine appears to destroy the function of the proline sandwich in theantibody, similar to glycine. The strongly reduced ADCC observed herefor the P329G mutant most likely resulted from the strongly reducedbinding to the FcγRIIA and FcγRIIIA receptor (see FIG. 1 e and FIG. 1 e).

Example 5

ADCC with Target Cells

S Human peripheral blood mononuclear cells (PBMC) were used as effectorcells and were prepared using Histopaque-1077 (Sigma Diagnostics Inc.,St. Louis, MO63178 USA) and following essentially the manufacturer'sinstructions. In brief, venous blood was taken with heparinized syringesfrom volunteers. The blood was diluted 1:0.75-1.3 with PBS (notcontaining Ca++ or Mg++) and layered on Histopaque-1077. The gradientwas centrifuged at 400×g for 30 min at room temperature (RT) withoutbreaks. The interphase containing the PBMC was collected and washed withPBS (50 ml per cells from two gradients) and harvested by centrifugationat 300×g for 10 minutes at RT. After resuspension of the pellet withPBS, the PBMC were counted and washed a second time by centrifugation at200×g for 10 minutes at RT. The cells were then resuspended in theappropriate medium for the subsequent procedures. The effector to targetratio used for the ADCC assays was 25:1 and 10:1 for PBMC and NK cells,respectively. The effector cells were prepared in AIM-V medium at theappropriate concentration in order to add 50 ml per well of round bottom96 well plates. Target cells were 20 human B lymphoma cells (e.g., Rajicells) grown in DMEM containing10% FCS. Target cells were washed in PBS,counted and resuspended in AIM-V at 0.3 million per ml in order to add30'000 cells in 100 ml per microwell. Antibodies were diluted in AIM-V,added in 50 ml to the pre-plated target cells and allowed to bind to thetargets for 10 minutes at RT. Then the effector cells were added and theplate was incubated for 4 hours at 37″° C. in a humified atmospherecontaining 5% CO₂. Killing of target cells was assessed by measurementof lactate dehydrogenase (LDH) release from damaged cells using theCytotoxicity Detection kit (Roche Diagnostics, Rotkreuz, Switzerland).After the 4-hour incubation the plates were centrifuged at 800×g. 100 mlsupernatant from each well was transferred to a new transparent flatbottom 96 well plate. 100 ml color substrate buffer from the kit wereadded per well. The V_(max) values of the color reaction were determinedin an ELISA reader at 490 nm for at least 10 min using SOFTmax PROsoftware (Molecular Devices, Sunnyvale, CA94089, USA), Spontaneous LDHrelease was measured from wells containing only target and effectorcells but no antibodies. Maximal release was determined from wellscontaining only target cells and 1% Triton X-100. Percentage of specificantibody-mediated killing was calculated as follows:((x−SR)/(MR−SR)*100, where x is the mean of Vmax at a specific antibodyconcentration, SR is the mean of Vmax of the spontaneous release and MRis the mean of V_(max) of the maximal release.

The potency to recruit immune-effector cells depends on type of Fcvariant as measured by classical ADCC assay. Here, buman NK92 cell-linetransfected with human FegRIIIA was used as effector and CD20 positiveRaji cells were used as target cells. As can be seen in FIG. 4 a theADCC is strongly reduced in GA101 (CD20) Fc variants wherein glycinereplaces proline (P329G) and also, to a similar extent, in the doublemutant P329G/LALA. In contrast the ADCC decrease was less strong withthe LALA mutation. In order to better distinguish between the differentvariants, the variants were also produced in the glycoengineered versionto enhance the ADCC potential. It can be observed that the parentalmolecule (GA101 (CD20)) shows strong ADCC as expected. The LALA versionis strongly impaired in its ADCC potential, The P3290 mutant verystrongly decreased the ADCC; much more than a P329A variant of the GA101(CD20) antibody (FIG. 4 b ).

Example 6

Complement Activity

Target cells were counted, washed with PBS, resuspended in AIM-V(Invitrogen) at I million cells per ml. 50 ml cells were plated per wellin a flat bottom. 96 well plate. Antibody dilutions were prepared inAIM-V and added in 50 ml to the cells. Antibodies were allowed to bindto the cells for 10 minutes at room temperature. Human serum complement(Quidel) was freshly thawed, diluted 3-fold with AIM-V and added in 50ml to the wells. Rabbit complement (Cedarlane Laboratories) was preparedas described by the manufacturer, diluted 3-fold with AIM-V and added in50 ml to the wells. As a control, complement sources were heated for 30min at 56° C. before addition to the assay. The assay plates wereincubated for 2h at 37° C. Killing of cells was determined by measuringLDH release. Briefly, the plates were centrifuged at 300×g for 3 min. 50ml supernatant per well were transferred to a new 96 well plate and 50ml of the assay reagent from the Cytotoxicity Kit (Roche) were added. Akinetic measurement with the ELISA reader determined the Vmaxcorresponding with LDH concentration in the supernatant. Maximal releasewas determined by incubating the cells in presence of 1% Triton X-100.

The different Fc variants were analyzed to mediate CDC on SUDH-14 targetcells. The non-glycoengineered GA101 molecule shows clear induction ofCDC. The LALA variant shows activity only at the highest concentration,whereas and the P3290 and P329G/LALA variants do not show any CDCactivity (FIG. 5 a ). Moreover, the LALA variant as well as the P329Gand P329A variants of a glycoengineered GA101 molecule do not show anyCDC activity (FIG. 5 b ).

Example 7

Carbohydrate Profile of Human IgG1

The carbohydrate profiles of human IgGI antibodies containing mutationswithin the Fe, aimed at abrogating the binding to Fcγreceptors, wereanalyzed by MALDI/TOF-MS in positive ion mode (neutraloligosaccharides).

Human (h) IgG1 variants were treated with sialidase (QA-Bio) followingthe manufacturer's instructions to remove terminal sialic acid. Theneutral oligosaccharides of hIgG1 were subsequently released by PNGase F(QA-Bio) digestion as previously described (Ferrara, C. et al., Biotech.Bioeng. 93 (2006) 851-861). The carbohydrate profiles were analyzed bymass spectrometry (Autoflex, Bruker Daltonics GmbH) in positive ion modeas previously described (Ferrara, C. et al., Biotech, Bioeng. 93 (2006)851-861).

The carbohydrate profile of the neutral Fe-associated glycans of humanIgGI is characterized by three major m/z peaks, which can be assigned tofucosylated. complex oligosaccharide with none (G0), one (G1) or two(G2) terminal galactose residues.

The carbohydrate profiles of hlg01 containing mutations within the Fo,aimed at abrogating binding to Fo receptors, were analyzed and comparedto that obtained for the wild type antibody. The IgG variants containingone of the mutations within the Fc (P329G, LALA, P329A, P329G/LALA) showsimilar carbohydrate profiles to the wild type antibody, with theFc-associated glycans being fucosylated complex oligosaccharides (datanot shown). Mutation within the Fc can affect the level of terminalgalactosylation and terminal sialylation, as observed by replacing aminoacid at positions 241, 243, 263, 265, or 301 by alanine (Lund, J. etal., J. Immunol. 157 (1996) 4963-4969).

FIG. 6 a shows the relative percentage of galactosylation for thedifferent hIgG1 Fc-variants described here. Slight variations can beobserved when the antibodies are expressed in a different host, but nosignificant difference in terminal galactosylation could be observed.

FIG. 6 b indicates the variability in galactosylation content for wildtype and IgG1-P329G/LALA for 4 different antibodies, where fourdifferent V-domains were compared for their amount of galactosylationwhen expressed in Hek293 EBNA cells.

Example 8

Antibody-Induced Platelet Aggregation in Whole Blood Assay.

Whole blood platelet aggregation analysis using the Multiplateinstrument from Dynabyte. First, 20 ml blood from normal human donorsare withdrawn and transferred into hiruidin tubes (Dynabyte Medical, #MP0601). Plug minicell Impedance device (Dynabead #MP0021) into theMultiplate instrument was used for the assay. Then, 175 μl 0.9% NaClwere added to the minicell. Antibody was added to minicell to obtain thefinal test concentration. Then, 175 μl homan blood were added andincubated for 3 min at 37ºC, Automated start of impedance analysis foradditional 6 min at 37° C. The data were analyzed by quantification ofarea-under-the-curve as a measure of platelet aggregation.

The CD9 antibody has been shown to induce platelet activation andplatelet aggregation (Worthington, et al., Br. J. Hematol. 74(2) (1990)216-222). Platelet aggregation induced by antibodies binding toplatelets previously has been shown to involve binding to FcγRIIA (deReys, et al., Blood 81 (1993) 1792-1800). As shown above the mutationsLALA, P329G, P329G/LALA and P329G/SPLE introduced into the CD9 antibodystrongly reduced binding of the CD9 antibody to the FcγRIIA receptor(FIG. 1 e ).

The activation (measured by Ca efflux, data not shown) as well asplatelet aggregation induced by & CD9 antibody was eliminated byintroducing the P329G and LALA triple mutation into the antibody suchthat the FcγRIIA binding is strongly reduced compared to the wildtypeantibody (see FIGS. 7 a and 7 b ). Murine IgGI induced plateletaggregation at low antibody concentrations (0.1-1 μ/ml). At higherconcentrations overstimulation of platelets leads to silencing of theaggregation response (3-30 ng/ml). Donor variability was observed withchim-hu-IgG4-SPLE. In FIG. 6 a data for a chim-hu-IgG4-SPLE responder athigher antibody concentrations and in FIG. 6 b data for achim-bu-IgG4-SPLE non-responder is shown. None of the blood samplesshowed any aggregation response with the antibody variantschim-hu-IgG1-LALA, chim-hu-IgG-WT-P329G, chim-hu-IgG1-LALA-P329G,chim-hu-OgG4-SPLE-P329G, chim-hu-IgG4-SPLE-N297Q. Controls: spontaneousaggregation in untreated blood sample (background); ADP-induced (ADP)and Thrombin analogon-induced (TRAP6) platelet aggregation. Isotypecontrols: Murine IgGI (murine Isotype) and human IgG4-SPLE (hu-IgG4-SPLEIsotype).

One possible interpretation of these data is that the decreased bindingof the CD9 antibody with the triple mutations to the FcγRIIA receptor isthe reason for the diminished platelet aggregation observed with thesekind of mutant antibodies. In principle, prevention of thrombocyteaggregation, as a toxic side-effect of an antibody treatment, might thusbe possible by introducing the above mentioned mutations, capable ofreducing binding to the FcγRIIA receptor, into the Fc part of anantibody.

1. A polypeptide comprising an Fc variant of a wild-type human IgG Fcregion, said Fc variant comprising an amino acid substitution atposition Pro329 and at least one further amino acid substitution,wherein the residues are numbered according to the EU index of Kabat,and wherein said polypeptide exhibits a reduced affinity to the humanFcγRIDA and/or FcγRIIA and/or FcγRI compared to a polypeptide comprisingthe wildtype IgG Fc rogion, and wherein the ADCC induced by saidpolypeptide is reduced to at least 20% of the ADCC induced by thepolypeptide comprising a wild-type human IgG Fc region.
 2. Thepolypeptide according to claim 1, wherein Pro329 of a wild-type human Fcregion is substituted with glycine or arginine or an amino acid residuelarge enough to destroy the proline sandwich within the Fe/Fcγreceptorinterface.
 3. The polypeptide according to any one of claim 1 or 2,wherein said at least one further amino acid substitution is S228P,E233P, L234A, L235A, L235E, N297A, N297D, or P331S.
 4. The polypeptideaccording to any one of claims 1-3, wherein said at least one furtheramino acid substitution is L234A and L235A of the human IgGI Fc regionor S228P and 1235E of the human IgG4 Fc region.
 5. The polypeptideaccording to any one of claims 1-4, wherein the affinity to at least onefurther receptor of the group comprising the human receptors FcγI,FcγIIA, and C1q is reduced compared to the polypeptide comprising awild-type human IgG Fc region.
 6. The polypeptide according to any oneof claims 1-5, wherein the polypeptide comprises a human IgGI or IgG4 Fcregion.
 7. The polypeptide according to any one of claims 1-6, whereinthe polypeptide is an antibody or an Fc fusion protein.
 8. Thepolypeptide according to any one of claims 1-7, wherein thrombocyteaggregation induced by the polypeptide is reduced compared to thethrombocyte aggregation induced by a polypeptide comprising a wild-typehuman IgG Fc region.
 9. The polypeptide according to any one of claims1-8, wherein CDC induced by the polypeptide is strongly reduced comparedto the CDC induced by a polypeptide comprising a wild-type human IgG Fcregion.
 10. The polypeptide according to any one of claims 1-9 for useas a medicament.
 11. The polypeptide according to any one of claims1-10, wherein the polypeptide is an anti-CD9 antibody, which ischaracterized in that the polypeptide comprising the wildtype Fc regioncomprises as heavy chain variable region SEQ ID NO:9 and as variablelight chain region SEQ ID NO:8.
 12. The polypeptide according to any oneof claims 1-11, for use in treating a disease wherein it is favorablethat an effector function of the polypeptide is strongly reducedcompared to the effector function induced by a polypeptide comprising awild-type human IgG Fc region.
 13. Use of the polypeptide according toany one of claims 1-12 in the manufacture of a medicament for thetreatment of a disease, wherein it is favorable that the effectorfunction of the polypeptide comprising an Fc variant of a wild-typehuman IgG Fc region is strongly reduced compared to the effectorfunction induced by a polypeptide comprising a wild-type human IgG Fcregion.
 14. A method of treating an individual having a disease, whereinit is favorable that the effector function of the polypeptide comprisingan Fc variant of a wild-type human IgG Fc region is strongly reducedcompared to the effector function induced by a polypeptide comprising awildtype human Fc polypeptide, comprising administering to an individualan effective amount of the polypeptide according to any one of claims1-12.
 15. Use of a polypeptide comprising an Fc variant of a wild-typehuman IgG Fc region, said polypeptide having Pro329 of the human IgG Fcregion substituted with glycine, wherein the residues are numberedaccording to the EU index of Kabat, wherein said polypeptide exhibits areduced affinity to the human FcγRIIIA and FcγRIA for down-modulation ofADCC to at least 20% of the ADCC induced by the polypeptide comprisingthe wildtype human IgG Fc region, and/or for down-modulation of ADCP.16. Use of a polypeptide according to claim 15, wherein the Fc variantcomprises at least two further amino acid substitution at L234A andL235A of the human IgGI Fc region or S228P and L235B of the human IgG4Fc region.
 17. The use according to claims 15-16, wherein thrombocyteaggregation induced by the polypeptide is reduced compared to thethrombocyte aggregation induced by a polypeptide comprising a wildtypehuman Fc region, wherein the polypeptide is a platelet activatingantibody.
 18. A method of treating an individual having a disease with apolypeptide, said polypeptide having Pro329 of the human IgG Fc regionsubstituted with Gly, wherein the residues are numbered according to theEU index of Kabat, wherein said polypeptide is characterized by astrongly reduced binding to FcγRIIIA and FcγRIIA compared to apolypeptide comprising a wildtype human IgG Fc region, comprisingadministering to the individual an effective amount of said polypeptide.19. The method according to claim 18, wherein said polypeptide comprisesat least two further amino acid substitution at L234A and L235A of thehuman IgG1 Fc region or S228P and L235E of the human IgG4 Fc region.